CAEV-based vector systems

ABSTRACT

This invention relates to caprine arthritis encephalitis virus-based vectors and vector systems that are useful in the delivery of nucleic acids to both non-dividing and dividing cells. Methods for delivering nucleic acids to both non-dividing and dividing cells using the vector systems are also disclosed.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to lentiviral vectors useful in polynucleotidedelivery, and more specifically to caprine arthritis encephalitisvirus-based vectors useful in polynucleotide delivery to non-dividingand dividing cells.

2. Related Art

Lentiviruses are a subgroup of retroviruses that are capable ofinfecting non-dividing, as well as dividing cells. Vectors derived fromlentiviruses are ideal tools for delivering exogenous genes to targetcells because of their ability to stably integrate into the genome ofdividing and non-dividing cells and to mediate long-term gene expression(Gilbert and Wong-Staal, 2001; Mitrophanous et al., 1999; Naldini etal., 1996; Sauter and Gasmi, 2001).

Lentiviruses have been isolated from many vertebrate species includingprimates, e.g., human and simian immunodeficiency viruses (HIV-1, HIV-2,SIV), as well as non-primates, e.g., feline immunodeficiency virus(FIV), bovine immunodeficiency virus (BIV), equine infectious virus(EIAV), caprine arthritis encephalitis virus (CAEV) and the visna virus.Of these, HIV and SIV are presently best understood. However, use ofsuch systems in humans raises serious safety concerns, due to thepossibility of recombination by the vector into a virulent anddisease-causing form. Accordingly, non-primate lentiviruses arepreferred for use in gene therapy.

Among non-primate lentiviral vectors, vectors derived from FIV (Curranand Nolan, 2002) and EIAV [US 2001/0044149] are best characterized, andlittle progress has been made for other non-primate lentiviral vectors.

CAEV, like all lentiviruses, can infect and replicate in dividing cellsas well as in terminally differentiated and non-dividing cells. Severalfeatures of CAEV biology make this virus an attractive candidate todevelop into a gene transfer/therapy vector. First, the normal host ofCAEV is goats, and there are no reported cases of human infection byCAEV. Second, the CAEV genome is phylogenetically most distant fromHIV-1 among lentiviruses. Third, the genome organization of the CAEV isrelatively simple compared with other lentiviruses. The CAEV genomecontains three structural genes (gag, pol, env) and threeregulatory/accessory genes (vif, tat and rev).

Despite these advantages, however, efforts to develop CAEV-baseddelivery systems have not been successful, resulting only in unsafe andinefficient recombinant viral vector production systems, rendering theuse of CAEV-based gene delivery systems impractical.

In 1998, L. Mselli-Lakhal et al. reported on the first generationCAEV-based vector system, but the viral titers of the system (i.e.,10-187 TU/ml) were below useful levels. The authors attributed theinefficiency to a lack of accumulation of genomic RNA into thecytoplasm, and the low packaging efficiency of the vector RNA. Anothershortcoming of the study was the use of an infectious wild-type virus(“helper virus”) as its packaging system, which is of little practicalvalue in human applications.

Accordingly, a need remains for a safe and efficient CAEV-basedlentiviral vector system capable of mediating gene transfer into a broadrange of dividing and non-dividing cells.

SUMMARY OF THE INVENTION

The present invention is broadly directed to the production ofCAEV-based lentiviral vector particles useful for delivering exogenouspolynucleotides into target cells. These vector particles find use inanti-viral, anti-tumor and/or gene therapies.

The present invention provides in one aspect a transfer vector for usein a CAEV-based vector production system described herein, the transfervector comprises (a) a CAEV packaging sequence consisting essentially of(i) the untranslated region between the CAEV 5′ LTR and the CAEVgag-encoding sequence, and (ii) nucleotides 1 to X of the CAEVgag-encoding sequence linked to the 3′ end of said untranslated region,wherein X is less than 613, and (b) cis-acting elements required forpolyadenylation, RNA transport, reverse transcription, and integration,in operable association with said packaging sequence.

In one embodiment of the invention, X is selected from the groupconsisting of: 60, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325,350, 375, 400, 425, 450, 475, 500, 525, 550, 575 and 600.

In another embodiment of the invention, X is selected from the groupconsisting of:

-   -   X is greater than 25 and less than 600,    -   X is greater than 25 and less than 500,    -   X is greater than 25 and less than 400,    -   X is greater than 25 and less than 300,    -   X is greater than 25 and less than 200,    -   X is greater than 50 and less than 600,    -   X is greater than 50 and less than 500,    -   X is greater than 50 and less than 400,    -   X is greater than 50 and less than 300,    -   X is greater than 50 and less than 200,    -   X is greater than 75 and less than 600,    -   X is greater than 75 and less than 500,    -   X is greater than 75 and less than 400,    -   X is greater than 75 and less than 300,    -   X is greater than 75 and less than 200,    -   X is greater than 100 and less than 600,    -   X is greater than 100 and less than 500,    -   X is greater than 100 and less than 400,    -   X is greater than 100 and less than 300,    -   X is greater than 100 and less than 200,    -   X is greater than 125 and less than 600,    -   X is greater than 125 and less than 500,    -   X is greater than 125 and less than 400,    -   X is greater than 125 and less than 300,    -   X is greater than 125 and less than 200,    -   X is greater than 150 and less than 600,    -   X is greater than 150 and less than 500,    -   X is greater than 150 and less than 400,    -   X is greater than 150 and less than 300,    -   X is greater than 150 and less than 200,    -   X is greater than 200 and less than 600,    -   X is greater than 200 and less than 500,    -   X is greater than 200 and less than 400,    -   X is greater than 200 and less than 300,    -   X is greater than 200 and less than 200,    -   X is greater than 250 and less than 600,    -   X is greater than 250 and less than 500,    -   X is greater than 250 and less than 400, and    -   X is greater than 250 and less than 300.

In another embodiment, X is greater than 40 and less than 613.

In another embodiment, X is greater than 57 and less than 613.

In yet another embodiment, X is about 327.

In one embodiment of the invention, the start codon of the gag-encodingsequence is mutated to prevent translation of gag protein. In a furtherembodiment, the start codon is mutated to TAG.

In another embodiment of the transfer vector of the invention, the ATGcodon of the gag-encoding sequence is located X base pairs downstream ofthe start codon ATG, wherein start codon is mutated to preventtranslation of gag protein, and wherein X is less than 30. In a furtherembodiment X is about 21.

The transfer vector of the invention may further comprise an RRE region.

In another embodiment of the invention, the transfer vector comprisesthe CAEV 3′ LTR wherein the U3 region is deleted.

The transfer vector of the present invention may further comprise aheterologous promoter. In one embodiment of the invention, theheterologous promoter is the human cytomegalovirus major immediate earlypromoter (HCMV MIEP). In a further embodiment, the transfer vector ispCAH/SINd1 (SEQ ID NO: 68).

The transfer vector of the present invention may further comprise atranscription cassette comprising a heterologous polynucleotide ofinterest operably linked to a heterologous promoter (e.g., humancytomegalovirus major immediate-early promoter HCMV MIEP, or murinecytomegalovirus major immediate-early promoter MCMV MIEP). Such atransfer vector permits the incorporation of the polynucleotide ofinterest into virus particles, thereby providing a means for amplifyingthe number of infected host cells containing the polynucleotide therein.

The present invention also provides a CAEV-based lentiviral vectorsystem for producing CAEV-based, replication-defective vector particlesuseful in delivering exogenous polynucleotides into mammalian cells. Thevector particles are capable of infecting and transducing mammaliancells. The vector system comprises the transfer vector described above,and a packaging vector system, wherein said packaging vector systemcomprises: a first polynucleotide comprising a CAEV gag-pol-encodingsequence and an RRE, and a second polynucleotide comprising a viralenvelope encoding sequence.

In one embodiment, the second polynucleotide comprises a non-CAEVenv-encoding sequence. In one embodiment the second polynucleotidecomprises a VSV-G- or GaLV-encoding sequence.

In another embodiment, the CAEV vector system comprises a thirdpolynucleotide sequence comprising a rev-encoding sequence.

In another embodiment, the CAEV vector system comprises a fourthpolynucleotide sequence comprising a vif-encoding sequence.

In a further embodiment, the first polynucleotide of each of the CAEVvector systems described above further comprises a heterologousregulatory sequence operably linked to the CAEV gag-pol-encodingsequence.

In a further embodiment, the second polynucleotide of theabove-described CAEV vector systems further comprises a heterologousregulatory sequence operable linked to said viral envelope-encodingsequence.

In a further embodiment, the third polynucleotide further comprises aheterologous regulatory sequence operably linked to the rev-encodingsequence.

In a further embodiment, the fourth polynucleotide further comprises aheterologous regulatory sequence operably linked to the vif-encodingsequence.

In one embodiment of the invention, the CAEV vector system comprises apackaging vector system which is devoid of a competent CAEV packagingsequence. In a further embodiment, the packaging vector system is devoidof the 5′ end of the CAEV genome between the splice donor site and thegag start codon.

In one embodiment, the CAEV vector system comprises a first vectorcomprising the first polynucleotide and a second vector comprising thesecond polynucleotide. In another embodiment, the vector systemcomprises a first vector comprising the first polynucleotide, a secondvector comprising the second polynucleotide, and a third vectorcomprising the third polynucleotide. In another embodiment, the vectorsystem comprises a first vector comprising the first polynucleotide, asecond vector comprising the second polynucleotide, a third vectorcomprising the third polynucleotide, and a fourth vector comprising thefourth polynucleotide. The third vector may be pHYK/rev (SEQ ID NO: 75),and the fourth vector may be pHYK/vif (SEQ ID NO: 76).

In yet another embodiment, the vector system comprises a first vectorcomprising the first polynucleotide, the third polynucleotide and thefourth polynucleotide, and a second vector comprising the secondpolynucleotide.

In one embodiment, the first vector of the CAEV vector system comprisesa CAEV gag-encoding sequence and an RRE operable linked to aheterologous promoter. The promoter may be an MCMV MIEP. In a furtherembodiment, the CAEV vector system comprises the first vector pMGP/RRE(SEQ ID NO: 77).

In one embodiment, the second vector of the CAEV vector system is aVSV-G-encoding sequence operably linked to a heterologous promoter. Thepromoter may be an HCMV MIEP. The second vector may further comprise abeta globin intron. In a further embodiment, the CAEV vector systemcomprises the second vector pHGVSV-G (SEQ ID NO: 74).

In one embodiment, the second vector of the CAEV vector system is a GaLVenv-encoding sequence operably linked to a heterologous promoter. Thepromoter may be an MCMV MIEP. The second vector may further comprise aeukaryotic elongation factor-1 alpha intron. In a further embodiment,the CAEV vector system comprises the second vector pMYKEF-1/env (SEQ IDNO: 72).

Another aspect of the invention is a method of producing a CAEV-basedlentiviral vector particle useful for infecting mammalian cells. Themethod comprises (a) transfecting a cell with the vector systemdescribed supra, under conditions suitable for production of CAEV-basedparticles, where the vector particle is infection- andtransduction-competent, and replication-defective, and (b) recoveringthe vector particle.

The present invention also provides a composition comprising aCAEV-based lentiviral vector particle and optionally a carrier, wherethe vector particle is produced by the methods described supra.

The present invention also provides a kit comprising the transfer vectoror the CAEV-based lentiviral vector system described supra.

The present invention also provides a packaging cell comprising a CAEVgag-pol-encoding sequence and RRE, and optionally a viral env-encodingsequence. The packaging cell may further comprise a rev-encoding and/ora vif-encoding sequence. The cell is useful for packaging the RNA formof the transfer vector into an infection- and transduction-competentvector particle, which is replication-defective.

In one embodiment, the vector system comprises a cell comprising thefirst polynucleotide described supra. The vector system may furthercomprise the third and/or the fourth polynucleotide described supra.

In another embodiment, the vector system comprises a cell comprising thefirst polynucleotide and second polynucleotides described supra. Thevector system may further comprise the third and/or the fourthpolynucleotide described supra.

In another embodiment, the vector system comprises a cell comprising afirst vector that comprises a CAEV gag-pol-encoding sequence and an RRE.The first vector may further comprise a rev-encoding and/or avif-encoding sequence. Alternatively, the cell may comprise a firstvector comprising a CAEV gag-pol-encoding sequence and an RRE, a secondvector comprising a rev-encoding sequence and/or a third vectorcomprising a vif-encoding sequence.

In some embodiments, the vector system comprises a cell comprising afirst vector that comprises a CAEV gag-pol-encoding sequence and an RRE,and a second vector that comprises a viral env-encoding sequence. Thefirst vector may further comprise a rev-encoding and/or a vif-encodingsequence. Alternatively, the cell may comprise a first vector comprisingthe CAEV gag-pol-encoding sequence and an RRE, a second vectorcomprising a viral env-encoding sequence, and optionally a third vectorcomprising a rev-encoding sequence and/or a fourth vector comprising avif-encoding sequence.

Another aspect of the present invention is a method of delivering apolynucleotide or polypeptide into a mammalian cell or replicating apolynucleotide molecule encoding said polypeptide, comprising contactinga mammalian cell with the vector particle described supra underconditions which may allow for integration of said polynucleotide intothe genome of said cell and optionally under conditions allowingexpansion of said polypeptide encoded by said polynucleotide. Themammalian cell may be a dividing cell, a non-dividing cell or a CD34+stem cell. The method of delivering a polynucleotide or a polypeptideinto a mammalian cell or replicating a polynucleotide molecule encodingsaid polypeptide, may further comprise isolating the cell from a mammalprior to contacting the cell with the vector particle. The method mayfurther comprise expanding said cell in culture after contacting it withthe vector particle. The method may further comprise reintroducing thecell into a mammal before or after expanding the contacted cell.

The present invention further provides a method for delivering apolypeptide into a vertebrate, comprising administering to thevertebrate a CAEV-based lentiviral vector particle comprising aheterologous polynucleotide of interest, where the vector particle isproduced by the method described supra, such that the polypeptideencoded by the delivered polynucleotide is expressed in the vertebrate,in an amount sufficient to be detectable or to elicit a biologicalresponse in the vertebrate.

The present invention further provides a vector comprising a CAEVpackaging sequence consisting essentially of (a) the untranslated regionbetween the CAEV 5′ LTR and the CAEV gag-encoding sequence, and (b)nucleotides 1 to X of the CAEV gag-encoding sequence linked to the 3′end of the untranslated region, wherein X is less than 613.

The inventors have discovered that the production of the CAEV-basedlentiviral vector particles, as described herein, results in enhancedefficiency and safety in the lentiviral vector design over the existingCAEV-based vector particles. The enhanced efficiency is achieved throughthe discovery of the optimal length of the untranslated region betweenthe 5′LTR and the gag start codon and the gag-encoding region, whichserves as an efficient packaging sequence by allowing efficientencapsidation, which then results in increased viral titers. Viral titeris also improved by using a strong heterologous promoter in the designof the packaging plasmids. The enhanced safety is achieved through theconstruction of a tat-independent transfer vector and a plasmid-basedpackaging system.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 is a schematic illustration of the CAEV proviral genomicorganization.

FIG. 2A is a schematic illustration of the plasmid pMGP/RRE (SEQ ID NO:77). pMGP/RRE (SEQ ID NO: 77) is a 9,446 bp plasmid which contains anMCMV MIEP region (located at bp 1-660) located upstream of the CAEVgag-pol coding region (bp 709-5,243), the RRE region (5,426-5,627 or bp5,368-5,669), and the bovine growth hormone (BGH) polyadenylation signal(bp 5,751-5,984). The vector also contains a neomycin resistance genecoding region (bp 8,151-7,155), a SV40 origin of replication (bp8,509-8,152), a Col E1 origin of replication (bp 6,115-6,698), and anampicillin resistance gene region (bp 9,362-8,528).

FIG. 2B is a schematic illustration of the plasmid pMGP/REV/RRE.pMGP/REV/RRE is a 9,924 bp plasmid which contains an MCMV MIEP region(located at bp 1-660) and the major splicing donor of CAEV (bp 688-704)located upstream of the CAEV gag-pol coding region (bp 726-5,258), thefirst exon rev coding region (bp 5,383-5,494), the RRE region (bp5,540-5,841), the second exon rev coding region (bp 5,888-6,177), andthe bovine growth hormone (BGH) polyadenylation signal (bp 6,229-6,462).The vector also contains a neomycin resistance gene coding region (bp7,633-8,629), a SV40 origin of replication (bp 8,987-8,630), a Col E1origin of replication (bp 6,593-7,176), and an ampicillin resistancegene region (bp 9,840-9,006).

FIG. 3A is a schematic illustration of the plasmid pCAH/SINd (SEQ ID NO:73). pCAH/SINd (SEQ ID NO: 73) is a 3,566 bp plasmid which contains theHCMV MIEP (bp 1-588), the R-U5 sequence regions in the CAEV 5′LTR (bp611-772), the RRE region (bp 796-1,154), and the U3-deleted CAEV 3′LTRregion (bp 1,275-1,458). The vector also contains a Col E1 origin ofreplication (bp 1,863-2,466), and a kanamycin resistance gene codingregion (bp 2,698-3,510).

FIG. 3B is a schematic illustration of the plasmid pCAH/SINd0 (SEQ IDNO: 67). pCAH/SINd0 (SEQ ID NO: 67) is a 3,911 bp plasmid which containsthe HCMV MIEP (bp 1-588), the R-U5 sequence regions in CAEV 5′LTR (bp611-772), the residual untranslated sequences containing the primerbinding site (PBS) (bp 773-789), the RRE region (bp 1,141-1,499), andthe U3-deleted CAEV 3′LTR region (bp 1,620-1,803). The vector alsocontains a Col E1 origin of replication (bp 2,208-2,791) and a kanamycinresistance gene coding region (bp 3,043-3,855).

FIG. 3C is a schematic illustration of the plasmid pCAH/SINd1 (SEQ IDNO: 68). pCAH/SINd1 (SEQ ID NO: 68) is a 4,238 bp plasmid which containsthe HCMV MIEP (bp 1-588) promoter, the R-U5 sequence regions in the CAEV5′LTR (bp 610-772), the residual untranslated sequences containing thePBS site (bp 773-789), the 327 bp fragment of the gag gene (bp1,121-1,448) with ATG to TAG point mutations at the start ATG codon(bp1121-1123) and the ATG codon (bp1142-1144) located downstream of thestart ATG codon, the RRE region (bp 1,468-1,826) and the U3-deleted CAEV3′LTR region (bp 1,947-2,130). The vector also contains a Col E1 originof replication (bp 2,535-3,118) and a kanamycin resistance gene region(bp 3,370-4,182).

FIG. 3D is a schematic illustration of the plasmid pCAH/SINd2 (SEQ IDNO: 69). Plasmid pCAH/SINd2 (SEQ ID NO: 69) is a 4,523 bp plasmid whichcontains the HCMV MIEP (bp 1-588), the R-U5 sequence regions in the CAEV5′LTR (bp 610-772), the residual untranslated sequences containing thePBS site (bp 773-789), the 612 bp fragment of the gag gene (bp1,121-1,733), with point mutations at the start ATG codon (bp 1121-1123)and the ATG codon (bp 1142-1144) located downstream of the start ATGcodon, the RRE region (bp 1,753-2,111) and the U3-deleted CAEV 3′LTRregion (bp 2,232-2,415). The vector also contains a Col E1 origin ofreplication (bp 2,820-3,403) and a kanamycin resistance gene codingregion (bp 3,655-4,467).

FIG. 3E is a schematic illustration of the plasmid pCAH/SINd3 (SEQ IDNO: 70). pCAH/SINd3 (SEQ ID NO: 70) is a 4,819 bp plasmid which containsthe HCMV MIEP (bp 1-588), the R-U5 sequence regions in CAEV 5′LTR (bp610-772), the residual untranslated sequences containing PBS site (bp773-789), the 908 bp fragment of the gag gene (bp 1,121-2,029) withpoint mutations at the start ATG codon (bp 1121-1123) and the ATG codon(bp 1142-1144) located downstream of the start ATG codon, the RRE region(bp 2,049-2,407) and the U3-deleted CAEV 3′LTR region (bp 2,549-2,711).The vector also contains a Col E1 origin of replication (bp 3,116-3,699)and a kanamycin resistance gene coding region (bp 3,951-4,763).

FIG. 3F is a schematic illustration of the plasmid pCAH/SINd4 (SEQ IDNO: 71). pCAH/SINd4 (SEQ ID NO: 71) is a 5,112 bp plasmid which containsthe HCMV MIEP (bp 1-588), the R-U5 sequence regions in the CAEV 5′LTR(bp 610-772), the residual untranslated sequences containing the PBSsite (bp 773-1,120), the 1198 bp fragment of the gag gene (bp1,121-2,319) with point mutations at the start ATG codon (bp 1121-1123)and the ATG codon (bp 1142-1144) located downstream of the start ATGcodon, the RRE region (bp 2,342-2,700) and the U3-deleted CAEV 3′LTRregion (bp 2,842-3,004). The vector also contains a Col E1 origin ofreplication (bp 3,409-3,992), and a kanamycin resistance gene codingregion (bp 4,244-5,056).

FIG. 3G is a schematic illustration of the plasmid pCAH/SINd1/hlacZ (SEQID NO: 79). pCAH/SINd1/hlacZ (SEQ ID NO: 79) is an 8,127 bp plasmidderived from the pCAH/SINd1 (SEQ ID NO: 68) that expresses the lacZreporter gene. The vector contains two HCMV MIEP promoter regions(located at bp 1-588 and bp 1,866-2,460, respectively), the R-U5sequence regions in the CAEV 5′LTR (bp 610-772), the residualuntranslated sequences containing the PBS site (bp 773-789), the 325 bpfragment of gag gene (bp 1,121-1,446) with point mutations at the startATG codon (bp 1121-1123) and the ATG codon (bp 1142-1144) locateddownstream of the start ATG codon, the RRE region (bp 1,466-1,836), thelacZ gene coding sequence (bp 2,541-5,711), and the U3-deleted CAEV3′LTR region (bp 5,782-6,019). The vector also contains a Col E1 originof replication (bp 6,424-7,007), and a kanamycin resistance gene codingregion (bp 7,259-8,071).

FIG. 3H is a schematic illustration of the plasmid pCAH/SINd60/hlacZ(SEQ ID NO: 78). Plasmid pCAH/SINd60/hlacZ (SEQ ID NO: 78) is a 7,856 bpwhich contains two promoter regions, HCMV MIEP (located at bp 1-588 andbp 1,595-2,189, respectively), the R-U5 sequence regions in the CAEV5′LTR (bp 610-772), the residual untranslated sequences containing thePBS site (bp 773-789 bp), the 60 bp fragment of gag gene (bp1,121-1,181) with point mutations at the start ATG codon (bp 1121-1123)and the ATG codon (bp 1142-1144) located downstream of the start ATGcodon, the RRE region (bp 1,195-1,565), the lacZ gene coding sequence(bp 2,270-5,440), and the U3-deleted CAEV 3′LTR region (bp 5,511-5,748).The vector also contains a Col E1 origin of replication (bp6,153-6,736), and a kanamycin resistance gene coding region (bp6,988-7,800).

FIG. 4 is a schematic illustration of the plasmid pHYK/vif (SEQ ID NO:76). pHYK/vif (SEQ ID NO: 76) is a 5,729 bp plasmid which contains theHCMV MIEP (bp 1-596), the vif gene coding region (bp 691-1,380), the BGHpolyadenylation signal (bp 1,467-1,695), a Col E1 origin of replication(bp 1,826-2,409), a neomycin resistance gene coding region (bp3,862-2,866), and an ampicillin resistance gene coding region (bp5,270-4,239).

FIG. 5 is a schematic illustration of the plasmid pHYK/rev (SEQ ID NO:75). pHYK/rev (SEQ ID NO: 75) is a 5,419 bp plasmid which contains theHCMV MIEP (bp 1-596), the rev gene coding region (bp 672-1,073), the BGHpolyadenylation signal (bp 1,157-1,385), a Col E1 origin of replication(bp 1,516-2,099), a neomycin resistance gene coding region (bp3,552-2,556), and an ampicillin resistance gene coding region (bp4,960-3,929).

FIG. 6A is a schematic illustration of the plasmid pHGVSV-G (SEQ ID NO:74). pHGVSV-G (SEQ ID NO: 74) is a 7,623 bp plasmid which contains theHCMV MIEP (bp 1-596), the β-globin intron region (bp 714-1,599), theVSV-G coding region (bp 1,632-3,312), the BGH polyadenylation signal (bp3,361-3,589), a Col E1 origin of replication (bp 3,720-4,303), aneomycin resistance gene coding region (bp 5,756-4,760), an ampicillinresistance gene coding region (bp 7,164-6,133), and a F1 origin ofreplication (bp 7,165-7,621).

FIG. 6B is a schematic illustration of the plasmid pMYKEF1/env (SEQ IDNO: 72). pMYKEF1/env (SEQ ID NO: 72) is a 7,579 bp plasmid whichcontains the MCMV MIEP (bp 1-665), a human EF1-α intron region (bp668-1,618), the GaLV env coding region (bp 1,699-3701), the BGHpolyadenylation signal (bp 3,885-4,118), a Col E1 origin of replication(bp 4,349-4,832), a neomycin resistance gene coding region (bp6,290-5,284), and an ampicillin resistance gene coding region (bp7,496-6,666).

FIG. 7 shows a photograph illustrating the relative amount of transfervector RNA transcribed from gene transfer vectors transfected into human293T target cells.

FIG. 8 shows two photographs illustrating gene transfer into human 293Ttarget cells by CAEV (A) and MuLV (B) vectors.

FIG. 9 shows a photographic illustration of the relative amount oftransfer vector RNA expressed in the transfected 293T cells (lanes 1, 2and 3), and encapsidated in and released from the 293T packaging cells(lanes 4, 5 and 6).

FIG. 10 shows a photograph illustrating the relative amount of transfervector RNA encapsidated in and released from human 293T packaging cells.FIG. 11 shows a photograph illustrating the relative amount ofintegrated retroviral cDNA after infection and reverse transcription oflentiviral vectors pseudotyped by VSV-G or GaLV envelope protein.

FIG. 12 shows a photograph illustrating the relative amount of viralvector cDNA integrated into the infected host cell chromosome.

FIG. 13 shows two graphs illustrating the FACS analysis of (A) thecontrol cells, and (B) the G1-arrested cells.

FIG. 14 shows two graphs illustrating (A) the number of transduced cellsand (B) the relative transduction efficiencies of HIV-1-, CAEV-, andMuLV-derived viral vectors on dividing and non-dividing cells.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to, inter alia, CAEV-based lentiviral vectorsystems and methods employing said vectors to deliver polypeptides ofinterest into dividing and non-dividing cells.

The CAEV Genome

The wild-type CAEV virus has a dimeric RNA genome (single-stranded,positive polarity) that is replicated through a double-stranded DNAintermediates and is packaged into a spherical enveloped virioncontaining a nucleoprotein core. The genome contains three genes thatencode the structural and enzymatic proteins Gag, Pol, and Env, and longterminal repeats (LTR) at each end of the integrated viral genome. Inaddition, the genome encodes three regulatory proteins, vif, tat, andrev.

The gag gene encodes the internal structural proteins, the pol geneencodes viral replication enzymes, and the env gene encodes an envelopeglycoprotein that mediates attachment of virus to the cell surface. TheVif protein is associated with viral infectivity, and the Tat proteinwith transactivation of the 5′ LTR. The Rev protein and its targetsequence RRE (Rev responsive element) are associated with the stabilityof viral RNA, regulation of viral RNA splicing, and transport of largeRNA (unspliced and singly-spliced) from the nucleus to the cytoplasm.The proviral LTR sequences contain the U3 (unique sequence elementlocated downstream from the structural proteins), R (short repeat ateach end of the genome), and U5 (unique sequence element immediatelyafter the R sequence) regions. The U3 region of 5′LTR contains the viralpromoter and enhancers. The 3′ end of the genome containspolyadenylation signal in the 3′LTR.

The wild-type genome of CAEV also contains several cis-acting elements,including atts (attachment site) at the end of LTRs for provirusintegration); promoter elements that control transcriptional initiationof the integrated provirus at the 5′LTR; a PBS (primer binding site)located downstream of the 5′LTR; a 5′-splice donor site; a packagingsequence (herein referred to interchangeably as a packaging site or apackaging signal); a ppt (polypurine tract) site located near the 3′LTR;and polyadenylation signals at the 3′LTR.

As used herein, the term “cis” is used in reference to the presence ofgenes on the same chromosome or linear portion of a nucleic acid.Therefore, the term “cis-defect” refers to a defect found on a linearsequence of a nucleic acid. The term “cis-acting” is used in referenceto the controlling effect of a regulatory gene on a gene present on thesame chromosome or linear portion of a nucleic acid. For example,promoters, which affect the synthesis of downstream mRNA are cis-actingcontrol elements.

The complete genomic sequence for two isolates of CAEV are known and thesequences are deposited in the National Center for BiotechnologyInformation (NCBI) database as NC_(—)001463 (SEQ ID NO: 1) and AF322109(SEQ ID NO: 2) (Saltarelli et al., 1990, and Gjerset, B. J. et al.unpublished, respectively). The nucleic acids of the claimed inventionare not limited to a particular isolate of CAEV, but rather to asequence that retains the known function of that genomic sequence. Forexample, it is known in the art that natural variations in a genesequence may occur during viral replication, resulting in a similarnucleic acid sequence that encodes proteins having a similar function.

A sequence alignment of the NC_(—)001463 (SEQ ID NO: 1) and AF322109(SEQ ID NO: 2) genomic sequences is shown in TABLE 1. As is visible inTABLE 1, there is considerable nucleic acid identity between thesequences, however differences at the nucleic acid level are apparent.Of particular importance is the variability of the CAEV gag regiondenoted in TABLE 2 (SEQ ID NOs: 3-6). Sequence alignments ofNC_(—)001463 5′LTR, pol, rev, and vif genes and the corresponding genesfrom AF322109 can be found in TABLES 3-6 (SEQ ID NOs: 7-14),respectively. Many partial sequences of the CAEV genome are also knownand have been deposited. For example, accession numbers AY081139,AY101347, AY101348, AY047362, AF402668, AF402667, AF402666, AF402665,AF402664, AJ305042, AJ305041, and AJ305040 all provide for sequences ofthe gag gene from Brazilian isolates of CAEV. Accession numbersAF015181, L78453, L78451, L78450, L78447, and L78446 also contain thesequences of gag genes from a variety of CAEV isolates. Accessionnumbers X64828 and M63106 contain the sequences of rev genes from avariety of CAEV isolates. Accession numbers AF015182, AJ305053, K03327,L78448, L78452 and U35814 contain pol genes from a variety of CAEVisolates. A sequence alignment between the NC_(—)001463 gag gene (SEQ IDNOs: 15, 17) and the AF015181 gag gene (SEQ ID NOs: 16, 18) is found inTABLE 7. A sequence alignment between the NC_(—)001463 gag gene (SEQ IDNOs: 19, 25) and the gag genes from AF402664 (SEQ ID NOs: 20, 26),AF402665 (SEQ ID NOs: 21, 27), AF402666 (SEQ ID NOs: 22, 28), AF402667(SEQ ID NOs: 23, 29), AF402668 (SEQ ID NOs: 24, 30) is found in TABLE 8.A sequence alignment between the NC_(—)001463 gag gene (SEQ ID NOs: 31,35) and the gag genes from AJ305040 (SEQ ID NOs: 32, 36), AJ305041 (SEQID NOs: 33, 37), AJ305042 (SEQ ID NOs: 34, 38) is found in TABLE 9. Asequence alignment between the NC_(—)001463 gag gene (SEQ ID NOs: 39,41) and the gag gene from AY047362 (SEQ ID NOs: 40, 42) is found inTABLE 10. A sequence alignment between the NC_(—)001463 (SEQ ID NOs: 43,45) gag gene and the gag gene from AY081139 (SEQ ID NOs: 44, 46) isfound in TABLE 11. A sequence alignment between the NC_(—)001463 (SEQ IDNOs: 47, 50) gag gene and the gag genes from AY101347 (SEQ ID NOs: 48,51) and AY101348 (SEQ ID NOs: 49, 52) is found in TABLE 12. A sequencealignment between the NC_(—)001463 gag gene (SEQ ID NOs: 53, 59) and thegag genes from L78446 (SEQ ID NOs: 54, 60), L78447 (SEQ ID NOs: 55, 61),L78450 (SEQ ID NOs: 56, 62), L78451 (SEQ ID NOs: 57, 63), and L78453(SEQ ID NOs: 58, 64) is found in TABLE 13.

The alignments were performed using VectorNTI (Informax, USA) using thefollowing parameters:

For pairwise alignment: gap opening penalty: 15

-   -   Gap extension penalty: 6.6

For multiple alignment: gap opening penalty: 15

-   -   Gap extension penalty : 6.6    -   Gap separation penalty range : 8

TABLE 14 is a summary of the percent identity values for the sequencealignments of gag gene sequences listed above. TABLE 15 is a summary ofthe percent identity of the full genomic alignment, and alignments ofthe gag, 5′ LTR, pol, rev, and vif regions of NC_(—)001463 (SEQ IDNO: 1) and AF322109 (SEQ ID NO: 2). Given that the genomic sequence oftwo CAEV isolates, in addition to a large number of partial sequencesfrom a variety of CAEV isolates are known and consensus sequences can beeasily discerned, it would not require undue experimentation to practicethe claimed invention using a variety of CAEV sequences.

CAEV Vectors of the Invention

The vectors of the present invention provide a means for replicating andexpressing polynucleotides or genes independent of the host cell nucleusin a broad phylogenetic range of host cells. This vector-mediatedincorporation of heterologous nucleic acid into a host cell is referredto as transfection or infection of the host cell, wherein infectionmeans the use of virus particles, and transfection means the use ofnaked molecules of nucleic acid.

The term “gene” refers to a DNA sequence that comprises control andcoding sequences necessary for the production of a polypeptide orprecursor. The term “polynucleotide” or “nucleic acid molecule”, as usedinterchangeably herein, refers to nucleotide polymers of any length,such as two or more, and includes both DNA and RNA. The nucleotides canbe deoxyribonucleotides, ribonucleotides, nucleotide analogs (includingmodified phosphate moieties, bases, or sugars), or any substrate thatcan be incorporated into a polymer by a suitable enzyme, such as a DNApolymerase or an RNA polymerase. The polypeptide can be encoded by afull-length coding sequence or by any portion of the coding sequence solong as the desired activity of the polypeptide is retained.

The term “wild-type” refers to a gene or gene product which has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the term“modified” or “mutant” refers to a gene or gene product which displaysmodifications in sequence and or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product.Naturally-occurring mutants can be isolated, and are identified by thefact that they have altered characteristics when compared to thewild-type gene or gene product.

It must be noted that as used in this specification and the appendedclaims, the singular forms “a”, “an”, “the”, and the like, includeplural references unless the context clearly dictates otherwise. Thus,for example, reference to “a polynucleotide” includes polynucleotidesand “a stem cell” includes a plurality of cells.

As used herein, the term “retrovirus” is used in reference to RNAviruses that utilize reverse transcriptase during their replicationcycle. The retroviral genomic RNA is converted into double-stranded DNAby reverse transcriptase. This double-stranded DNA form of the virus iscapable of being integrated into the chromosome of the infected cell;once integrated, it is referred to as a “provirus.” The provirus servesas a template for RNA polymerase II and directs the expression of RNAmolecules which encode the structural proteins and enzymes needed toproduce new viral particles.

As used herein, the term “lentivirus” refers to a group (or genus) ofretroviruses that give rise to slowly developing disease. Virusesincluded within this group include human immunodeficiency virus (HIV);visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) insheep, caprine arthritis encephalitis virus (CAEV); equine infectiousanemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunedeficiency virus (BIV); and simian immunodeficiency virus (SIV).Diseases caused by these viruses are characterized by a long incubationperiod and protracted course. Usually, the viruses latently infectmonocytes and macrophages, from which they spread to other cells.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer polynucleotide (e.g. DNA) segments from one cellto another. The term “vehicle” is sometimes used interchangeably with“vector.” It is intended that any form of vehicle or vector beencompassed within this definition. For example, vectors include, butare not limited to viral particles, plasmids, transposons, etc.

Standard techniques for the construction of the vectors of the presentinvention are well-known to those of ordinary skill in the art and canbe found in such references as Sambrook et al., Molecular Cloning: ALaboratory Manual 2nd Ed. (Cold Spring Harbor, N.Y., 1989). A variety ofstrategies are available for ligating fragments of DNA, the choice ofwhich depends on the nature of the termini of the DNA fragments andwhich choices can be readily made by the skilled artisan.

Suitable polyadenylation sequences of the present invention include, butare not limited to the bovine growth hormone (BGH) polyadenylationsignal (Pfarr et al., 1986), the SV40 early region polyadenylation site(Hall et al., 1983) and the SV40 late region polyadenylation site(Carswell and Alwine, 1989), β-globin polyA, and herpes simplex virusthymidine kinase polyA.

A promoter of the present invention may comprise a promoter of mammalianor viral origin, and will be sufficient to direct the transcription of adistally located sequence (i.e. a sequence linked to the 5′ end of thepromoter sequence) in a cell. The promoter region may also includecontrol elements for the enhancement or repression of transcription.Suitable promoters include, but are not limited to, the human or murinecytomegalovirus immediate-early promoter (HCMV MIEP or MCMV MIEP),elongation factor 1 alpha (ef-1α), and Rous Sarcoma virus long terminalrepeat promoter (pRSV). Intron sequences may also be combined with apromoter. Intron sequences include, but are not limited to ef-1α intronand β-globin intron. Inducible expression systems may also be used.Examples of inducible systems include, but are not limited toecdysone-inducible mammalian expression system (Invitrogen, CA, USA) andTet-On and Tet-Off gene expression systems (Clontech, CA, USA). Cell ortissue specific promoters can be utilized to target expression of genesequences in specific cell populations.

Enhancer sequences upstream from the promoter or terminator sequencesand downstream of the coding region may be optionally included in thevectors of the present invention to facilitate expression. Vectors ofthe present invention may also contain additional nucleic acidsequences, such as an intron sequence, a localization sequence, or asignal sequence, sufficient to permit a cell to efficiently andeffectively process the protein expressed by the nucleic acid of thevector. Examples of intron sequences include the β-globin intron (Kim etal., 2002) and the human EF-1α intron (Kim et al., 2002). Suchadditional sequences are inserted into the vector such that they areoperably linked with the promoter sequence, if transcription is desired,or additionally with the initiation and processing sequence iftranslation and processing are desired. Alternatively, the insertedsequences may be placed at any position in the vector.

The term “operably linked” is used to describe a linkage between a genesequence and a promoter or other regulatory or processing sequence suchthat the transcription of the gene sequence is directed by an operablylinked promoter sequence, the translation of the gene sequence isdirected by an operably linked translational regulatory sequence, andthe post-translational processing of the gene sequence is directed by anoperably linked processing sequence.

The term “SIN vector” refers to the self-inactivating vector that has atruncated U3 region in the 3′ LTR. During reverse transcription, atruncated U3 is duplicated in the 5′LTR, resulting in the loss of thetranscription capacity and the interference effect on an internalpromoter.

The packaging sequence of the transfer vector consists essentially of(i) the untranslated region between the CAEV 5′ LTR and the CAEVgag-encoding sequence, and (ii) nucleotides 1 to X of the CAEVgag-encoding sequence linked to the 3′ end of said untranslated region,wherein X is less than 613. In one embodiment of the invention, X isselected from the group consisting of: 60, 100, 125, 150, 175, 200, 225,250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575 and600.

In another embodiment of the invention, X is selected from the groupconsisting of:

X is greater than 25 and less than 600,

X is greater than 25 and less than 500,

X is greater than 25 and less than 400,

X is greater than 25 and less than 300,

X is greater than 25 and less than 200,

X is greater than 50 and less than 600,

X is greater than 50 and less than 500,

X is greater than 50 and less than 400,

X is greater than 50 and less than 300,

X is greater than 50 and less than 200,

X is greater than 75 and less than 600,

X is greater than 75 and less than 500,

X is greater than 75 and less than 400,

X is greater than 75 and less than 300,

X is greater than 75 and less than 200,

X is greater than 100 and less than 600,

X is greater than 100 and less than 500,

X is greater than 100 and less than 400,

X is greater than 100 and less than 300,

X is greater than 100 and less than 200,

X is greater than 125 and less than 600,

X is greater than 125 and less than 500,

X is greater than 125 and less than 400,

X is greater than 125 and less than 300,

X is greater than 125 and less than 200,

X is greater than 150 and less than 600,

X is greater than 150 and less than 500,

X is greater than 150 and less than 400,

X is greater than 150 and less than 300,

X is greater than 150 and less than 200,

X is greater than 200 and less than 600,

X is greater than 200 and less than 500,

X is greater than 200 and less than 400,

X is greater than 200 and less than 300,

X is greater than 200 and less than 200,

X is greater than 250 and less than 600,

X is greater than 250 and less than 500,

X is greater than 250 and less than 400, and

X is greater than 250 and less than 300.

In another embodiment, X is greater than 40 and less than 613. In yetanother embodiment, X is about 327. In one embodiment of the transfervector, the codon which initiates gag translation has been mutated (e.g.ATG changed to TAG, TTG, CTG, or ATT) or deleted. The term “codon”refers to a sequence of three nucleotides in a DNA or messenger RNAmolecule that represents the instruction for incorporation of a specificamino acid into a growing polypeptide chain. The transfer vector furthercomprises a heterologous promoter and one or more cis-acting sequences.

As used herein, the term “packaging signal” or “packaging sequence”refers to sequences located adjacent to the 5′ LTR of the CAEV genomewhich are required for encapsidation of the viral RNA into the viralcapsid or particle. Several retroviral vectors use the minimal packagingsignal (also referred to as the psi [ψ] sequence) needed forencapsidation of the viral genome. Thus, as used herein, the terms“packaging sequence”, “packaging signal”, “psi”, and the symbol “ψ” areused in reference to the non-coding sequence required for encapsidationof CAEV RNA strands during viral particle formation.

In another embodiment of the invention, the transfer vector furthercomprises a transcription cassette. The term “transcription cassette” asused herein refers to a fragment or segment of nucleic acid containing aparticular grouping of genetic elements, generally a polynucleotidewhich expresses a polypeptide of interest, operably linked to aheterologous promoter. The cassette can be removed and inserted into avector or plasmid as a single unit.

An illustrative example of a transfer vector of the present invention isshown in FIG. 3C. FIG. 3C illustrates the plasmid pCAH/SINd1 (SEQ ID NO:68). PCAH/SINd1 (SEQ ID NO: 68) is a 4,238 bp plasmid that contains theHCMV MIEP promoter, the R-U5 sequence regions in the CAEV 5′LTR, theresidual untranslated sequences containing a PBS site, the 327 bpfragment of the gag gene with the ATG→TAG double point mutations, theRRE region and the U3-deleted CAEV 3′LTR region. The vector alsocontains a Col E1 origin of replication (bp 2535-3118) and a kanamycinresistance gene region (bp 3370-4182). The other illustrative examplesof transfer vectors are shown in FIG. 3A-3H.

The invention provides a CAEV vector system comprising the abovedescribed transfer vector and a packaging vector system. The packagingvector system comprises a first and second polynucleotide vectorsequence. The first polynucleotide sequence comprises CAEV gag-pol andRRE-encoding sequence and the second polynucleotide comprises a viralenvelope encoding sequence. In one embodiment, the second polynucleotideencodes a non-CAEV envelope.

The phrases “structural gene” as used herein refer to the polynucleotidesequence encode proteins which are required for encapsidation (e.g.,packaging) of the viral genome, and include gag, pol and env.

An illustrative example of a first packaging vector of the presentinvention is shown in FIG. 2A. FIG. 2A illustrates the plasmid pMGP/RRE(SEQ ID NO: 77). The plasmid contains 9,446 base pairs and includes aMCMV MIEP region, the CAEV gag-pol coding region, the RRE region, andthe bovine growth hormone (BGH) polyadenylation signal. The vector alsocontains a neomycin resistant gene coding region, a SV40 origin ofreplication, a Col E1 origin of replication, and an ampicillinresistance gene region.

It is possible to alter the host range of cells that the viral vectorsof the present invention can infect by utilizing an envelope gene fromanother closely related virus. In other words, it is possible to expandthe host range of the CAEV vectors of the present invention by takingadvantage of the capacity of the envelope proteins of certain viruses toparticipate in the encapsidation of other viruses. Examples ofretroviral-derived env gene include, but are not limited to: theG-protein of vesicular-stomatitis virus (VSV-G), gibbon ape leukemiavirus (GaLV), rous sarcoma virus (RSV), moloney murine leukemia virus(MoMuLV), mouse mammary tumor virus (MMTV), and human immunodeficiencyvirus (HIV). All of these viral envelope proteins efficiently formpseudotyped virions with genome and matrix components of other viruses.As used herein, the term “pseudotype” refers to a viral particle thatcontains nucleic acid of one virus but the envelope protein of anothervirus. In general, either VSV-G or GaLV pseudotyped vectors have a verybroad host range, and may be pelleted to titers of high concentration byultracentrifugation (Burns et al., 1993), while still retaining highlevels of infectivity.

Other illustrative examples of second packaging vectors of the presentinvention are shown in FIGS. 6A and 6B. FIG. 6A illustrates the plasmidpHGVSV-G (SEQ ID NO: 74). pHGVSV-G (SEQ ID NO: 74) is a 7,623 bp plasmidwhich contains the HCMV MIEP, the β-globin intron region, the VSV-Gcoding region, the BGH polyadenylation signal, a Col E1 origin ofreplication, a neomycin resistance gene coding region, an ampicillinresistance gene coding region, and an F1 origin of replication. FIG. 6Billustrates the plasmid pMYKEF1/env (SEQ ID NO: 72). This plasmidcontains 7,579 bp which includes the MCMV MIEP, a human EF1-α intronregion, the GaLV env coding region, the BGH polyadenylation signal, aCol E1 origin of replication, a neomycin resistance gene coding region,and an ampicillin resistance gene coding region.

In another embodiment of the invention, the packaging vector comprises athird polynucleotide which encodes Rev. In infected cells, Rev binds tothe Rev-responsive element (RRE) in viral transcripts and causes thetranscription of both singly-spliced and unspliced transcriptscharacteristic of the viral structural proteins in the late stage ofreplication. Accordingly, Rev mediates temporal regulation of viral geneexpression. Because mammalian cell splicing mechanisms are coupled totransport of mRNA from the site of synthesis in the nucleus to thecytoplasm, Rev also influences transport of viral transcripts containingRRE.

An illustrative example of a third packaging vector of the presentinvention is shown in FIG. 5. FIG. 5 illustrates the plasmid pHYK/rev(SEQ ID NO: 75). pHYK/rev (SEQ ID NO: 75) is a 5,419 bp plasmid whichcontains HCMV MIEP, the rev gene coding region, BGH polyadenylationsignal, a Col E1 origin of replication, a neomycin resistant gene codingregion, and an ampicillin resistant gene coding region.

In yet another embodiment of the invention, the packaging vectorcomprises a fourth polynucleotide encoding Vif. Incorporation of Vif maybe necessary for infection and packaging of virions, depending on thepackaging cell line chosen.

An illustrative example of a fourth packaging vector of the presentinvention is shown in FIG. 4. pHYK/vif (SEQ ID NO: 76) is a 5,729 bpplasmid which contains the HCMV MIEP, the vif gene coding region, theBGH polyadenylation signal, a Col E1 origin of replication, a neomycinresistance gene coding region, and an ampicillin resistance gene codingregion.

When retroviral vector DNA is transfected into the cells, it may or maynot become integrated into the chromosomal DNA and becomes transcribed,thereby producing full-length retroviral vector RNA that contains a ψsequence. Under these conditions, only the vector RNA is packaged intothe viral capsid structures. These complete, yet replication-defective,virus particles can then be used to deliver the retroviral vector totarget cells with relatively high efficiency.

As used herein, the term “replication-defective” refers to a virus thatis not capable of complete, effective replication such that infectivevirions are not produced (e.g. replication-defective lentiviralprogeny). The term “replication-competent” refers to wild-type virus ormutant virus that is capable of replication, such that viral replicationof the virus is capable of producing infective virions (e.g.,replication-competent lentiviral progeny).

It is also contemplated that packaging may be inducible, as well asnon-inducible. In inducible packaging cells and packaging cell lines,CAEV particles are produced in response to at least one inducer. Inpreferred embodiments with inducible cell lines, the inducer is Tat. Innon-inducible packaging cell lines and packaging cells, no inducer isrequired in order for lentiviral particle production to occur.

CAEV Vector Sequences

Functionally equivalent sequences of the present invention alsoencompass various fragments of a CAEV genome that retain substantiallythe same function as the respective native sequence. Such fragments willcomprise at least about 10, 15 contiguous nucleotides, at least about 20contiguous nucleotides, at least about 24, 50, 60, 80, 100, 120, 140,160, 180, 200, 220, 240, 260, 280, 300, 340, 360, 380, or up to theentire contiguous nucleotides of the specific genetic element ofinterest. Such fragments may be obtained by use of restriction enzymesto cleave the native viral genome; by synthesizing a nucleotide sequencefrom the native nucleotide sequence of the virus genome; or may beobtained through the use of PCR technology. See particularly (Mullis andFaloona, 1987) and (Erlich, 1989). Again, variants of the various vectorcomponents, such as those resulting from site-directed mutagenesis, areencompassed by the methods of the present invention. As described inmore detail below, methods are available in the art for determiningfunctional equivalence.

By “variant” it is intended to include substantially similar sequences.Thus, for nucleotide sequences or amino acid sequences, variants includesequences that are functionally equivalent to the various components ofthe viral vector system. Variant nucleotide sequences also includesynthetically derived nucleotide sequences that have been generated, forexample, by site directed mutagenesis, but which still retain thefunction of the native sequence. Generally, nucleotide sequence variantsor amino acid sequence variants of the invention will have at least 70%,generally 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to its respective native nucleotide sequence.

Variants of the invention include polynucleotides (e.g., vectors)comprising, consisting essentially of, or consisting of, sequences atleast 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the sequences of the vectors disclosed herein (SEQ ID NOs:67-79).

One of skill will appreciate that many conservative variations of thenucleic acid constructs disclosed yield a functionally identicalconstruct. Conservative variations of a particular nucleic acid sequencerefers to those nucleic acids which encode identical or essentiallyidentical amino acid sequences, or where the nucleic acid does notencode an amino acid sequence, to essentially identical sequences.Because of the degeneracy of the genetic code, a large number offunctionally identical nucleic acids encode any given polypeptide. Forexample, due to the degeneracy of the genetic code, “silentsubstitutions” (i.e., substitutions of a nucleic acid sequence which donot result in an alteration in an encoded polypeptide) are an impliedfeature of every nucleic acid sequence which encodes an amino acid.Similarly, “conservative amino acid substitutions,” in one or a fewamino acids in an amino acid sequence of a packaging or packageableconstruct are substituted with different amino acids with highly similarproperties, are also readily identified as being highly similar to adisclosed construct. For instance, the codons CGU, CGC, CGA, COG, AGA,and AGG all encode the amino acid arginine. Thus, at every positionwhere an arginine is specified by a codon, the codon can be altered toany of the corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of “conservatively modified variations.” Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation. One of skill will recognize that each codonin a nucleic acid (except AUG, which is ordinarily the only codon formethionine) can be modified to yield a functionally identical moleculeby standard techniques. Accordingly, each “silent variation” of anucleic acid which encodes a polypeptide is implicit in any describedsequence. Furthermore, one of skill will recognize that individualsubstitutions, deletions or additions which alter, add or delete asingle amino acid or a small percentage of amino acids (typically lessthan 5%, more typically less than 1%) in an encoded sequence are“conservatively modified variations” where the alterations result in thesubstitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. The following six groups each containamino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

See also, Creighton (1984) Proteins W. H. Freeman and Company. Finally,the addition of sequences which do not alter the activity of a nucleicacid molecule, such as a non-functional sequence is a conservativemodification of the basic nucleic acid. Such conservatively substitutedvariations of each disclosed sequence are a feature of the presentinvention.

With respect to the amino acid sequences for the various full-length ormature polypeptides used in the vector system of the present invention,variants include those polypeptides that are derived from the nativepolypeptides by deletion (so-called truncation) or addition of one ormore amino acids to the N-terminal and/or C-terminal end of the nativepolypeptide; deletion or addition of one or more amino acids at one ormore sites in the native polypeptide; or substitution of one or moreamino acids at one or more sites in the native polypeptide. Suchvariants may result from, for example, genetic polymorphism or fromhuman manipulation. Methods for such manipulations are generally knownin the art.

One of skill will recognize many ways of generating alterations in agiven nucleic acid construct. Such well-known methods includesite-directed mutagenesis, PCR amplification using degenerateoligonucleotides, exposure of cells containing the nucleic acid tomutagenic agents or radiation, chemical synthesis of a desiredoligonucleotide (e.g., in conjunction with ligation and/or cloning togenerate large nucleic acids) and other well-known techniques. See,(Gillam and Smith, 1979), (Roberts, Cheetham, and Rees, 1987), andSambrook, Innis, Ausbel, Berger, Needham VanDevanter and Mullis (allsupra).

A variant of a native nucleotide sequence or native polypeptide hassubstantial identity to the native sequence or native polypeptide. Avariant may differ by as few as 1 to 10 amino acid residues, such as6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue. Avariant of a nucleotide sequence may differ by as low as 1 to 30nucleotides, such as 6 to 20, as low as 5, as few as 4, 3, 2, or even 1nucleotide residue.

It is intended by “sequence identity” that the same nucleotides or aminoacid residues are found within the variant sequence and a referencesequence when a specified, contiguous segment of the nucleotide sequenceor amino acid sequence of the variant is aligned and compared to thenucleotide sequence or amino acid sequence of the reference sequence.Methods for sequence alignment and for determining identity betweensequences are well known in the art. With respect to optimal alignmentof two nucleotide sequences, the contiguous segment of the variantnucleotide sequence may have additional nucleotides or deletednucleotides with respect to the reference nucleotide sequence. Likewise,for purposes of optimal alignment of two amino acid sequences, thecontiguous segment of the variant amino acid sequence may haveadditional amino acid residues or deleted amino acid residues withrespect to the reference amino acid sequence. The contiguous segmentused for comparison to the reference nucleotide sequence or referenceamino acid sequence will comprise at least 20 contiguous nucleotides, oramino acid residues, and may be 30, 40, 50, 100, or more nucleotides oramino acid residues. Corrections for increased sequence identityassociated with inclusion of gaps in the variant's nucleotide sequenceor amino acid sequence can be made by assigning gap penalties.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. For example, percentidentity of an amino acid sequence can be determined using theSmith-Waterman homology search algorithm using an affine 6 gap searchwith a gap open penalty of 12 and a gap extension penalty of 2, BLOSUMmatrix 62. Alternatively, percent identity of a nucleotide sequence isdetermined using the Smith-Waterman homology search algorithm using agap open penalty of 25 and a gap extension penalty of 5. Such adetermination of sequence identity can be performed using, for example,the DeCypher Hardware Accelerator from TimeLogic Version G. TheSmith-Waterman homology search algorithm is taught in Smith andWaterman, herein incorporated by reference. Alternatively, the alignmentprogram GCG Gap (Wisconsin Genetic Computing Group, Suite Version 10.1)using the default parameters may be used. The GCG Gap program appliesthe Needleman and Wunch algorithm and for the alignment of nucleotidesequences with an open gap penalty of 3 and an extend gap penalty of 1may be used. Another preferred, non-limiting example of a mathematicalalgorithm utilized for the comparison of two sequences is the algorithmof Karlin and Altschul (Karlin and Altschul, 1990), modified as inKarlin and Altschul (Karlin and Altschul, 1993). Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al.(Altschul et al., 1990). BLAST nucleotide searches can be performed withthe NBLAST program, score=100, wordlength=12, to obtain nucleotidesequences having sufficient sequence identity. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3, toobtain amino acid sequences having sufficient sequence identity. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al. (Altschul et al., 1997).Alternatively, PSI-Blast can be used to perform an iterated search thatdetects distant relationships between molecules. See Altschul et al.(1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blastprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Anothernon-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers and Miller (1988)CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program(version 2.0), which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used. Percent identity of an amino acidsequence can also be determined using the VectorNTI (Informax, USA).

One of skill can select a desired nucleic acid of the invention basedupon the sequences provided and upon knowledge in the art regarding CAEVgenerally. The life-cycle, genomic organization, developmentalregulation and associated molecular biology of lentiviruses have beenthe focus of over a decade of intense research. The specific effects ofmany mutations in many lentiviral genomes are known. In addition, thenucleic acid sequence variations of some CAEV strains are known.Moreover, general knowledge regarding the nature of proteins and nucleicacids allows one of skill to select appropriate sequences with activitysimilar or equivalent to the nucleic acids and polypeptides disclosed inthe sequence listings herein.

Finally, most modifications to nucleic acids are evaluated by routinescreening techniques in suitable assays for the desired characteristic.For instance, changes in the immunological character of encodedpolypeptides can be detected by an appropriate immunological assay.Modifications of other properties such as nucleic acid hybridization toa complementary nucleic acid, redox or thermal stability of encodedproteins, hydrophobicity, susceptibility to proteolysis, or the tendencyto aggregate are all assayed according to standard techniques.

Polynucleotides of Interest

As will be appreciated by one skilled in the art, the nucleotidesequence of the inserted polynucleotide of interest may be of anynucleotide sequence. For example, the polynucleotide sequence may be areporter gene sequence or a selectable marker gene sequence. A reportergene sequence, as used herein, is any gene sequence which, whenexpressed, results in the production of a protein whose presence oractivity can be monitored. Examples of suitable reporter genes includethe gene for galactokinase, galactosidase, chloramphenicolacetyltransferase, β-lactamase, green fluorescent protein, enhancedgreen fluorescent protein, etc. Alternatively, the reporter genesequence may be any gene sequence whose expression produces a geneproduct that affects cell physiology. Polynucleotide sequences of thepresent invention may comprise one or more gene sequences that alreadypossess on or more promoters, initiation sequences, or processingsequences.

A selectable marker gene sequence is any gene sequence capable ofexpressing a protein whose presence permits one to selectively propagatea cell which contains it. Examples of selectable marker genes includegene sequences capable of conferring host resistance to antibiotics(e.g., puromycin, hygromycin, neomycin, zeocin and the like), or ofconferring host resistance to amino acid analogues, or of permitting thegrowth of bacteria on additional carbon sources or under otherwiseimpermissible culture conditions.

Reporter or selectable marker gene sequences are sufficient to permitthe recognition or selection of the vector in normal cells. In oneembodiment of the invention, the reporter gene sequence may encode anenzyme or other protein which is normally absent from mammalian cells,and whose presence can, therefore, definitively establish the presenceof the vector in such a cell.

The transfer vectors of the present invention additionally permit theincorporation of heterologous nucleic acid, or polynucleotides, intovirus particles, thereby providing a means for amplifying the number ofinfected host cells containing heterologous nucleic acid therein. Theincorporation of the heterologous polynucleotide facilitates thereplication of the heterologous nucleic acid within the viral particle,and the subsequent production of a heterologous protein therein. Aheterologous protein is herein defined as a protein or fragment thereofwherein all or a portion of the protein is not expressed by the hostcell. A nucleic acid or gene sequence is said to be heterologous if itis not naturally present in the wild-type of the viral vector used todeliver the gene into a cell. The term heterologous nucleic acidsequence or polynucleotide sequence, as used herein, is intended torefer to a nucleic acid molecule (preferably DNA). The polynucleotidesequence or heterologous polynucleotide sequence may also comprise thecoding sequence of a desired product such as a suitable biologicallyactive protein or polypeptide, immunogenic or antigenic protein orpolypeptide, or a therapeutically active protein or polypeptide. Thepolypeptide may supplement deficient or nonexistent expression of anendogenous protein in a host cell. Such gene sequences may be derivedfrom a variety of sources including DNA, cDNA, synthetic DNA, RNA orcombinations thereof. Such gene sequences may comprise genomic DNA whichmay or may not include naturally occurring introns. Moreover, suchgenomic DNA may be obtained in association with promoter sequences orpolyadenylation sequences. The gene sequences of the present inventionare preferably cDNA. Genomic or cDNA may be obtained in any number ofways. Genomic DNA can be extracted and purified from suitable cells bymeans well-known in the art. Alternatively, mRNA can be isolated from acell and used to prepare cDNA by reverse transcription, or other means.Alternatively, the polynucleotide sequence may comprise a sequencecomplementary to an RNA sequence, such as an antisense RNA sequence,which antisense sequence can be administered to an individual to inhibitexpression of a complementary polynucleotide in the cells of theindividual.

Expression of the heterologous gene may provide an immunogenic orantigenic protein or polypeptide to achieve an antibody response. Theantibodies thus raised may be collected from an animal in a body fluidsuch as blood, serum or ascites.

The heterologous gene can also be any nucleic acid of interest that canbe transcribed. Generally the foreign gene encodes a polypeptide.Preferably the polypeptide has some therapeutic benefit. The polypeptidemay supplement deficient or nonexistent expression of an endogenousprotein in a host cell. The polypeptide can confer new properties on thehost cell, such as a chimeric signaling receptor, see U.S. Pat. No.5,359,046. One of ordinary skill can determine the appropriateness of aforeign gene practicing techniques taught herein and known in the art.For example, the artisan would know whether a foreign gene is of asuitable size for encapsidation and whether the foreign gene product isexpressed properly.

The particular heterologous protein that can be employed in the presentinvention is not critical thereto.

Specific examples of such heterologous proteins which can be employed inthe present invention include dystrophin (Hoffman, Brown, and Kunkel,1987), coagulation factor VIII (Wion et al., 1985), Cystic FibrosisTransmembrane Regulator Protein (CFTR) (Anderson et al., 1991; Crawford,1991), Ornithine Transcarbamylase (OTC) (Murakami et al., 1988), andα1-antitrypsin (Fagerhol and Cox, 1981).

The genes encoding many heterologous proteins are well-known in the art,and can be cloned from genomic or cDNA libraries [Sambrook et al,supra]. Examples of such genes include the dystrophin gene (Lee et al.,1991), the Factor VIII gene (Toole et al., 1984), the CFTR gene (Rommenset al., 1989; Riordan, 1989), the OTC gene (Horwich et al., 1984), andthe α1-antitrypsin gene (Lemarchand et al., 1992).

In addition, genes encoding heterologous proteins such as Rb, for thetreatment of vascular proliferative disorders like atherosclerosis(Chang et al., 1995), and p53 for the treatment of cancer (Wills et al.,1994; Clayman, 1995), and HIV disease (Bridges and Sarver, 1995), can beemployed in the present invention.

The vector does not always need to code for a functional, heterologousgene product, i.e., it may also code for a partial gene product whichacts as an inhibitor of a eukaryotic enzyme (Warne, Viciana, andDownward, 1993; Wang, 1991).

It may also be desirable to modulate the expression of a gene regulatingmolecule in a cell by the introduction of a molecule by the method ofthe invention. The term “modulate” envisions the suppression ofexpression of a gene when it is over-expressed or augmentation ofexpression when it is under-expressed. Where a cell proliferativedisorder is associated with the expression of a gene, nucleic acidsequences that interfere with the expression of a gene at thetranslational level can be used. The approach can utilize, for example,antisense nucleic acid, ribozymes or triplex agents to blocktranscription or translation of a specific mRNA, either by masking thatmRNA with an antisense nucleic acid or triplex agent, or by cleavingsame with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules which are complementaryto at least a portion of a specific mRNA molecule . In the cell, theantisense nucleic acids hybridize to the corresponding mRNA forming adouble-stranded molecule. The antisense nucleic acids interfere with thetranslation of the mRNA since the cell will not translate an mRNA thatis double-stranded. Antisense oligomers of about 15 nucleotides or moreare preferred since such are synthesized easily and are less likely tocause problems than larger molecules when introduced into the targetcell. The use of antisense methods to inhibit the in vitro translationof genes is well known in the art (Marcus-Sekura, 1988).

The antisense nucleic acid can be used to block expression of a mutantprotein or a dominantly active gene product, such as amyloid precursorprotein that accumulates in Alzheimer's disease. Such methods are alsouseful for the treatment of Huntington's disease, hereditaryParkinsonism and other diseases. Antisense nucleic acids are also usefulfor the inhibition of expression of proteins associated with toxicity.

Use of an oligonucleotide to stall transcription can be by the mechanismknown as the triplex strategy since the oligomer winds arounddouble-helical DNA, forming a three-strand helix. Therefore, the triplexcompounds can be designed to recognize a unique site on a chosen gene(Maher, Wold, and Dervan, 1991; Helene, 1991).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode those RNA's, it is possible to engineer moleculesthat recognize and cleave specific nucleotide sequences in an RNAmolecule (Cech, 1988). A major advantage of that approach is only mRNA'swith particular sequences are inactivated.

It may be desirable to transfer a nucleic acid encoding a biologicalresponse modifier. Included in that category are immunopotentiatingagents including nucleic acids encoding a number of the cytokinesclassified as “interleukins”, for example, interleukins 1 through 12.Also included in that category, although not necessarily workingaccording to the same mechanism, are interferons, and in particulargamma interferon (γ-IFN), tumor necrosis factor (TNF) andgranulocyte-macrophage colony stimulating factor (GM-CSF). It may bedesirable to deliver such nucleic acids to bone marrow cells ormacrophages to treat inborn enzymatic deficiencies or immune defects.Nucleic acids encoding growth factors, toxic peptides, ligands,receptors or other physiologically important proteins also can beintroduced into specific non-dividing cells.

Thus, the recombinant CAEV vector system of the invention can be used totreat an HIV-infected cell (e.g., T-cell or macrophage) with an anti-HIVmolecule. In addition, respiratory epithelium, for example, can beinfected with a recombinant lentivirus of the invention having a genefor cystic fibrosis transmembrane conductance regulator (CFTR) fortreatment of cystic fibrosis.

Thus, the recombinant CAEV vector system of the invention can be used totreat many human diseases. Specific examples of possible application ofthe CAEV vector system in human diseases include, but are limited to:Alzheimer's diseases, Parkinson's diseases, amyotrophic lateralsclerosis disease, Huntington's disease, beta-thalassemia, retinitispigmentosa, mucopolysaccharide disease, leukodystrophy diseases,X-linked SCID, phenylketonuria, tryosinemia, hemophilia A and B,Wilson's diseases, LDL receptor deficiency, Human Immunodeficiency, andDuchenne's dystrophy.

CAEV Vector Particles

In a method of the invention, infectious and replication-defective CAEVvector particles may be prepared according to the methods disclosedherein in combination with techniques known to those skilled in the art.The method includes transfecting a lentivirus-permissive cell with thevector expression system of the present invention; producing theCAEV-derived particles in the transfected cell; and collecting the virusparticles from the cell.

The term “transfection” as used herein refers to the introduction offoreign DNA into eukaryotic cells. Transfection may be accomplished by avariety of means known to the art including but not limited to calciumphosphate-DNA co-precipitation, DEAE-dextran-mediated transfection,polybrene-mediated transfection, electroporation, microinjection,liposome fusion, lipofection and protoplast fusion. These techniques arewell known in the art.

As used herein, the term “transduction” refers to the delivery of a geneusing a viral or retroviral vector particles by means of infectionrather than by transfection. In some embodiments, retroviral vectors aretransduced. Thus, a “transduced gene” is a gene that has been introducedinto the cell via lentiviral or vector infection and provirusintegration. In certain embodiments, the CAEV viral vector particlestransduce genes into “target cells” or host cells.

The step of facilitating the production of the infectious viralparticles in the cells may also be carried out using conventionaltechniques, such as by standard cell culture growth techniques.

The step of collecting the infectious virus particles may also becarried out using conventional techniques. For example, the infectiousparticles may be collected by collection of the supernatant of the cellculture, as is known in the art. Optionally, the collected virusparticles may be purified if desired. Suitable purification techniquesare well known to those skilled in the art.

If desired by the skilled artisan, CAEV stock solutions may be preparedusing the vectors and methods of the present invention. Methods ofpreparing viral stock solutions are known in the art and are illustratedby, e.g., (Soneoka et al., 1995) and (Landau and Littman, 1992). In amethod of producing a stock solution in the present invention,lentiviral-permissive cells are transfected with the vector system ofthe present invention. The cells are then grown under suitable cellculture conditions, and the CAEV particles are collected from the cellculture media as described above. Suitable permissive cell linesinclude, but are not limited to, the human cell lines 293, 293T, andHeLa the monkey cell line Vero, and the goat cell lines GSM and Ch1Es.

The vectors of the present invention are also useful in preparing stablepackaging cells (i.e. cells that stably express CAEV structuralproteins, which cells, by themselves, cannot generate infectious virusparticles) and virus producer cells (VPC). Methods for preparingpackaging cells that express retrovirus proteins are known in the artand are exemplified by the methods set forth in, for example, U.S. Pat.No. 4,650,764 to Temin et al., which disclosure is incorporated hereinin its entirety. Within the scope of the present invention, a packagingcell will comprise a lentivirus-permissive host cell comprising a CAEVnucleic acid sequence from at least one CAEV packaging vector describedin this invention, which nucleic acid sequence is packaging-signaldefective, thus rendering the cell itself capable of producing at leastone CAEV structural protein, but not capable of producingreplication-competent infectious virus. A packaging cell may be made bytransfecting a CAEV-permissive host cell (e.g., a human embryonic kidney293 or 293T cells) with a suitable CAEV nucleic acid sequence asprovided above according to known procedures. The resulting packagingcell is thus able to express and produce at least one CAEV structuralprotein. However, the packaging cell is still not able to producerecombinant CAEV virus. The packaging cell may then be transfected withother nucleic acid sequences, i.e., a transfer vector, which may containheterologous genes of interest and an appropriate packaging signal. Oncetransfected with the additional sequence or sequences, the packagingcell may thus be used to provide stocks of CAEV viruses that containheterologous genes, but which viruses are themselvesreplication-incompetent. The resulting virus producing cell (VPC) isthus able to produce infectious virus particles containing heterologousgene of interest.

Gene Transfer and Therapy

A number of human genetic diseases that result from an alteration in asingle gene are prime candidates for gene therapy. As used herein, theterms “gene therapy” or “gene transfer” are defined as the insertion ofgenes into cells for the purpose of medicinal therapy. There are manyapplications of gene therapy, particularly via stem cell geneticinsertion, and thus are well known and have been extensively reviewed.The term “target” is used to indicate that the CAEV vector is intendedto transduce the cells. Target cells for therapeutic gene transfer,either ex vivo or in vivo, include, but are not limited to hematopoieticstem cell, lymphocyte, vascular endothelial cell, respiratory epithelialcell, keratinocyte, skeletal and muscle cells, liver cell, neuron cell,and cancer cell .

The gene transfer technology of the present invention may also be usedin elucidating the processing of peptides and identification of thefunctional domains of various proteins. Cloned cDNA or genomic sequencesfor proteins can be introduced into different target cells ex vivo, orin vivo, in order to study cell-specific differences in processing andcellular fate. By placing the coding sequences under the control of astrong promoter, a substantial amount of the desired protein can bemade. Furthermore, the specific residues involved in protein processing,intracellular sorting, or biological activity can be determined bymutational change in discrete residues of the coding sequences.

Gene transfer technology of the present invention can also be applied toprovide a means to control expression of a protein and to assess itscapacity to modulate cellular events. Some functions of proteins, suchas their role in differentiation, may be studied in tissue culture,whereas others will require reintroduction into in vivo systems atdifferent times in development in order to monitor changes in relevantproperties.

Gene transfer provides a means to study the nucleic acid sequences andcellular factors that regulate expression of specific genes. Oneapproach to such a study would be to fuse the regulatory elements to bestudied to reported genes and subsequently assaying the expression ofthe reporter gene.

Gene transfer also possesses substantial potential use in understandingand providing therapy for disease states. There are a number ofinherited diseases in which defective genes are known and have beencloned. In some cases, the function of these cloned genes is known. Ingeneral, the above disease states fall into two classes: deficiencystates, usually of enzymes, which are generally inherited in a recessivemanner, and unbalanced states, at least sometimes involving regulatoryor structural proteins, which are inherited in a dominant manner. Fordeficiency state diseases, gene transfer could be used to bring a normalgene into affected tissues for replacement therapy, as well as to createanimal models for the disease using antisense mutations. For unbalanceddisease states, gene transfer could be used to create a disease state ina model system, which could then be used in efforts to counteract thedisease state. Thus the methods of the present invention permit thetreatment of genetic diseases. As used herein, a disease state istreated by partially or wholly remedying the deficiency or imbalancewhich causes the disease or makes it more severe. The use ofsite-specific integration of nucleic sequences to cause mutations or tocorrect defects is also possible.

The method of the invention may also be useful for neuronal, glial,fibroblast or mesenchymal cell transplantation, or “grafting”, whichinvolves transplantation of cells infected with the recombinantlentivirus of the invention ex vivo, or infection in vivo into thecentral nervous system or into the ventricular cavities or subdurallyonto the surface of a host brain. Such methods for grafting will beknown to those skilled in the art and are described in Neural Graftingin the Mammalian CNS, Bjorklund & Stenevi, eds. (1985).

For diseases due to deficiency of a protein product, gene transfer couldintroduce a normal gene into the affected tissues for replacementtherapy, as well as to create animal models for the disease usingantisense mutations. For example, it may be desirable to insert a FactorVIII or IX encoding nucleic acid into a CAEV particle for infection of amuscle, spleen or liver cell.

There are many applications of gene therapy, particularly via stem cellgenetic insertion, and thus are well known and have been extensivelyreviewed. As used herein, the term “stem cells” includes but is notlimited to hematopoietic stem cells, neuronal stem cells, mesenchymal(particularly muscular) stem cells, and liver stem cells. Stem cells arecapable of repopulating tissues in vivo. Hematopoietic stem cells areprogenitor cells derived from primitive human hematopoietic cells.

Gene therapy using hematopoietic stem cells is also useful to treat agenetic abnormality in lymphoid and myeloid cells that results generallyin the production of a defective protein or abnormal levels ofexpression of the gene.

For a number of these diseases, the introduction of a normal copy orfunctional homolog of the defective gene and the production of evensmall amounts of the missing gene product would have a beneficialeffect. At the same time, overexpression of the gene product would notbe expected to have deleterious effects. The following provides anon-exhaustive list of diseases for which gene transfer intohematopoietic stem cells is potentially useful. These diseases generallyinclude bone marrow disorders, erythroid cell defects, metabolicdisorders and the like. Hematopoietic stem cell gene therapy isbeneficial for the treatment of genetic disorders of blood cells such asα- and β-thalassemia, sickle cell anemia and hemophilia A and B in whichthe globin gene or clotting factor genes (e.g., Factor IX and Factor Xgenes) are defective. Another good example is the treatment of severecombined immunodeficiency disease (SCIDS), in which patients lack theadenosine deaminase (ADA) enzyme which helps eliminate certainbyproducts that are toxic to T and B lymphocytes and render the patientsdefenseless against infection. Such patients are ideal candidates toreceive gene therapy by introducing the ADA gene into theirhematopoietic stem cells instead of the patient's lymphocytes as done inthe past. Other diseases include chronic granulomatosis where theneutrophils express a defective cytochrome b and Gaucher diseaseresulting from an abnormal glucocerebrosidase gene product inmacrophages.

Additionally, neurological degenerative disorder, e.g., Parkinson'sdisease, is an attractive target for gene therapy by introducing theGDNF (Glial cell line-derived neurotrophic factor) gene into thestriatum and the substantia (Kordower et al., 2000).

Strategies to treat various forms of cancer are also included in genetherapy. The CAEV vector can carry a gene that encodes, for example, atoxin or an apoptosis inducer effective to specifically kill thecancerous cells. Specific killing of tumor cells can also beaccomplished by introducing a suicide gene to cancerous hematopoieticcells under conditions that only the tumor cells express the suicidegene. The suicide gene product confers lethal sensitivity to the cellsby converting a normally nontoxic drug to a toxic derivative. Forexample, the enzyme cytosine deaminase converts the nontoxic substance5′-fluorocytosine to a toxic derivative, 5-fluorouracil (Mullen,Kilstrup, and Blaese, 1992). Tumor-specific lymphocytes can begenetically modified for example, to locally deliver gene products withanti-tumor activity to sites of the tumor to circumvent the toxicityassociated with the systemic delivery of these gene products. A genetherapy approach can also be applied to render bone marrow cellsresistant to the toxic effects of chemotherapy.

Gene therapy can also be used to prevent or combat viral infections suchas HIV and HTLV-1 infection. For example, hematopoietic stem cells canbe genetically modified to render them resistant to infection by HIV.One approach is to inhibit viral gene expression specifically by usingantisense RNA or by subverting existing viral regulatory pathways.Antisense RNAs complementary to retroviral RNAs have been shown toinhibit the replication of a number of retroviruses (To, Booth, andNeiman, 1986) including HIV (Rhodes and James, 1991) and HTLV-1 (vonRuden and Gilboa, 1989).

Another area where gene therapy in hematopoietic stem cells may find useis in alleviating autoimmune disease. The therapeutic gene can encode,e.g., a B or T cell signaling molecule capable of reconstituting thenormal apoptotic signal that results in the death and elimination ofautoreactive cells.

Ex vivo cell transformation for diagnostics, research, or for genetherapy (e.g., via re-infusion of the transformed cells into the hostorganism) is well known to those of skill in the art. In one embodimentof the invention, cells are isolated from the subject organism,transfected with a vector of the invention comprising a polypeptide ofinterest, and re-infused back into the subject organism (e.g., patient).

Various cell types suitable for ex vivo transformation are well known tothose of skill in the art. Particular preferred cells are stem cellsdescribed supra (see, e.g., Freshney (1994) Culture of Animal Cells, aManual of Basic Technique, third edition Wiley-Liss, New York, and thereferences cited therein for a discussion of how to isolate and culturecells from patients). Transformed cells are cultured by means well knownin the art. See, also Kuchler (1977) Biochemical Methods in Cell Cultureand Virology, Kuchler, R. J., Dowden, Hutchinson and Ross, Inc., andAtlas (1993) CRC Handbook of Microbiological Media (Parks ed) CRC press,Boca Raton, Fla. Mammalian cell systems often will be in the form ofmonolayers of cells, although mammalian cell suspensions are also used.Alternatively, cells can be derived from those stored in a cell bank(e.g., a blood bank). Illustrative examples of mammalian cell linesinclude the HEC-1-B cell line, VERO and Hela cells, Chinese hamsterovary (CHO) cell lines, W138, BHK, Cos-7 or MDCK cell lines (see, e.g.,Freshney, supra).

T cells or B cells are also used in some ex vivo gene transferprocedures. Several techniques are known for isolating T and B cells.The expression of surface markers facilitates identification andpurification of such cells.

In summary, the viral vectors of the present invention can be used tostably transduce either dividing or non-dividing cells, and stablyexpress a heterologous gene. Using this vector system, it is nowpossible to introduce into dividing or non-dividing cells, genes thatencode proteins that can affect the physiology of the cells. The vectorsof the present invention can thus be useful in gene therapy for diseasestates, or for experimental modification of cell physiology.

Kits

It is a further object of this invention to provide a kit or drugdelivery system comprising the vectors for use in the methods describedherein. All the essential materials and reagents required foradministration of the targeted retroviral particle may be assembled in akit (e.g., packaging cell construct or cell line). The components of thekit may be provided in a variety of formulations. The one or more CAEVparticles may be formulated with one or more agents (e.g., achemotherapeutic agent) into a single pharmaceutically acceptablecomposition or separate pharmaceutically acceptable compositions.

The components of these kits or drug delivery systems may also beprovided in dried or lyophilized forms. When reagents or components areprovided as a dried form, reconstitution generally is by the addition ofa suitable solvent, which may also be provided in another containermeans. The kits of the invention may also comprise instructionsregarding the dosage and or administration information for the targetedCAEV particle. The kits or drug delivery systems of the presentinvention also will typically include a means for containing the vialsin close confinement for commercial sale such as, e.g., injection orblow-molded plastic containers into which the desired vials areretained. Irrespective of the number or type of containers, the kits mayalso comprise, or be packaged with, an instrument for assisting with theinjection/administration or placement of the ultimate complexcomposition within the body of a subject. Such an instrument may be anapplicator, inhalant, syringe, pipette, forceps, measured spoon,eye-dropper or any such medically approved delivery vehicle.

The following examples illustrate various aspects of the invention, butin no way are intended to limit the scope thereof.

EXAMPLES

The following examples serve to illustrate certain embodiments andaspects of the present invention and are not to be construed as limitingthe scope thereof.

The following examples demonstrate the finding that the recombinantCAEV-based lentiviral vector system of the present invention is aseffective in expression as the well-known HIV-1 based lentiviral system.The examples show that the level of genomic RNA transcription,encapsidation, transduction, reverse transcription, and integration ofthe CAEV-based vector particle production system of the presentinvention is comparable to that of HIV-1-based lentiviral vector system,which has long been accepted as a highly efficient gene transfer system(Naldini et al., 1996).

This is the first report on the construction of a high titer CAEV-basedvector system, which is based on a minimum of three-plasmidco-transfection method, requiring the expression of just the gag-pol andenv genes, and optionally a rev gene.

Materials and Methods

Plasmid Construction

The Parent Plasmids.

The parent plasmids from which the CAEV vectors of the present inventionwere derived are the plasmid pWTE-BM and the plasmid pCAEV-LTR, kindlyprovided by Dr. Marie Suzan (Institut National de la Sante et de laRecherche Medicale “INSERM”, France) The pWTE-BM plasmid contains afull-length genomic CAEV cDNA except for the 0.4 kb Hind III fragmentwhich contains parts of env, rev, and U3 regions and a 1337 base pairstuffer fragment. The plasmid pCAEV-LTR contains the 0.4 kb Hind IIIfragment lacking in the pWTE-BM (Saltarelli et al., 1990; Saltarelli,1993). Neither of the vectors can generate a wild-type virus.

CAEV Gag-Pol Expression Vector (pMGP/RRE) (SEQ ID NO: 77).

The pMGP/RRE (SEQ ID NO: 77) plasmid is a PWTE-BM derived gag-polexpression plasmid (shown in FIG. 2A). The pMGP/RRE (SEQ ID NO: 77)plasmid contains a strong and heterologous MCMV major immediate-earlypromoter (MCMV MIEP), the gag-pol gene, and the rev responsive element(RRE). The pMGP/RRE (SEQ ID NO: 77) plasmid also encodes the neomycinresistant gene as an antibiotic selection marker. For the constructionof the plasmid, the gag-pol gene fragment (nucleotide 512 throughnucleotide 5046 of the CAEV genome) from pWTE-BM was subcloned into thepGL2-Basic (Promega, WI, USA) cloning vector by using standard protocolsfor several PCR and subcloning steps. The MCMV MIEP fragment was excisedfrom the plasmid pMYK (Kim et al., 2002) was then inserted upstream ofthe gag gene, and the RRE region (from nucleotide 7824 to nucleotide8183 or nucleotide 7849 to nucleotide 8150 of the CAEV genome) wasinserted downstream of the pol gene. The pMGP/REV/RRE is another gag-polexpressing plasmid (shown in FIG. 2B) containing the CAEV rev gene. Inaddition, the major splicing donor site of the CAEV (from nucleotide 330to nucleotide 346 of the CAEV genome) was inserted downstream of theMCMV promoter.

Transfer Vectors (pCAH/SINd Series).

The plasmids in the pCAH/SINd series (shown in FIGS. 3A-3H) (SEQ ID NOs:67-71, 73, 78, and 79) were constructed to identify an optimal packagingsequence for the design of the transfer vectors of the presentinvention. Each of the plasmids in the series were designed to containdifferent lengths of the 5′untranslated region and the beginning of thegag-encoding region to allow for the side by side comparison of theeffects of the various lengths in this region. To address certain safetyconcerns, these plasmids were designed as SIN (self-inactivation)vectors having the U3 region of the 3′LTR deleted. To allow high levelexpression of vector RNA from the transfer vectors in the absence of atrans-acting factor, tat, the U3 region of the 5′LTR was replaced withan HCMV MIEP. In addition, all known cis-acting sequence elementsrequired for polyadenylation, RNA transportation, reverse transcription,and integration were included in the transfer vector series.

The plasmids of the pCAH/SINd series (SEQ ID NOs: 67-71, 73, 78, 79)were constructed as follows. pCAH/SINd (PBS-deficient negative controlvector) (SEQ ID NO: 73) (FIG. 3A) was designed to contain only the 5′untranslated sequences (R and U5 region) in the 5′ LTR (from nucleotide1 to nucleotide 163 of the CAEV genome). pCAH/SINd0 (SEQ ID NO: 67)(FIG. 3B) was designed to contain the entire 5′ untranslated region(from nucleotide 1 to nucleotide 511 of the CAEV genome). pCAH/SINd1(SEQ ID NO: 68) (FIG. 3C) was designed to contain the entire 5′untranslated region and the 327 bp fragment of the gag gene (fromnucleotide 1 to nucleotide 839 of the CAEV genome) with point mutations.pCAH/SINd2 (SEQ ID NO: 69) (FIG. 3D) was designed to contain the entire5′ untranslated region and the 612 bp fragment of the gag gene (fromnucleotide 1 to nucleotide 1124 of the CAEV genome) with pointmutations. Plasmid pCAH/SINd3 (SEQ ID NO: 70) (FIG. 3E) was designed tocontain the entire 5′ untranslated region and the 908 bp fragment of thegag gene (from nucleotide 1 to nucleotide 1420 of the CAEV genome) withpoint mutations. Plasmid pCAH/SINd4 (SEQ ID NO: 71) (FIG. 3F) wasdesigned to contain the entire 5′ untranslated region and the 1,198 bpfragment of the gag gene (from nucleotide 1 to nucleotide 1710 of theCAEV genome) with point mutations. pCAH/SINd1/hlacZ (SEQ ID NO: 78)(FIG. 3G) was constructed by inserting the expression cassetteconsisting of the HCMV MIEP and the lacZ gene into the pCAH/SINd1 (SEQID NO: 68). The plasmid pCAH/SINd60/hlacZ (SEQ ID NO: 78) (FIG. 3H) hasthe same design as the pCAH/SINd1 (SEQ ID NO: 68) except for the lengthof the gag gene, where it contains the first 60 bp fragment of the gaggene with point mutations (from nucleotide 1 to nucleotide 569 of theCAEV genome).

CAEV Vif Expression Vector (pHYK/vif) (SEQ ID NO. 76).

The vif gene (from nucleotide 5006 to nucleotide 5695 of the CAEVgenome), which is known to be required for rapid and efficient virusreplication, was cloned into a eukaryotic expression vector pHYK (Kim etal., 2002) (FIG. 4).

CAEV Rev Expression Vector (pHYK/rev) (SEQ ID NO. 75).

The rev gene, which regulates viral gene expression at thepost-transcriptional level by interacting with the RRE, consists of twoexons (the first exon is positioned from nucleotide 6,012 to 6,123, andthe second exon is from nucleotide 8514 to 8803 of the CAEV genome). TheRev/RRE system promotes the nuclear export of unspliced RNA and is knownto be essential for lentiviral replication. The full-length cDNA of revgene was synthesized by RT-PCR and subcloned into the pHYK vector (FIG.5).

Viral Envelope Gene Expression Vector.

The envelope gene expression vector systems used herein are the plasmidpHGVSV-G (SEQ ID NO: 74) and the plasmid pMYKEF1/env (SEQ ID NO: 72)(FIGS. 6A and 6B). The plasmid pHGVSV-G (SEQ ID NO: 74) was designed toexpress the vesicular stomatitis virus G (VSV-G) glycoprotein andcontains the HCMV MIEP with β-globin intron as a promoter. ThepMYKEF-1/env (SEQ ID NO: 72) was designed to express the gibbon apeleukemia virus (GaLV) envelope protein and contains the MCMV MIEP witheukaryotic elongation factor-1α intron as a promoter.

MuLV- and HIV-1-Based Plasmids.

As control vector systems, pMFG/lac/Zpuro and pHR/lacZ vectors were usedin the present invention, that were lacZ-containing retrovirus vectorsderived from the murine leukemia virus (MuLV) (Kim et al., 1997) and thehuman immunodeficiency virus type 1 (HIV-1) (Naldini et al., 1996),respectively. For the packaging plasmids of MuLV and HIV-1 vectorsystems, pEQPAM3 (Persons et al., 1998) and pCMVΔR8-2 were used,respectively. The HIV-1 packaging plasmid pCMVΔR8-2 is identical withpCMVΔR9 (Naldini et al., 1996) except for encoding a functional HIV-1vpu gene and deletion of the 1.3-kb BglII fragment in env gene.

Vector Particle Production

Pseudotyped CAEV-based lentiviral vector particles were produced byliposome mediated transient transfection of three or more plasmids into293T cells plated one day prior to transfection at a density of 5×10⁵cells per 6-well culture dish. Three plasmid cotransfections wereperformed at a 1:1:1 molar ratio of a gag-pol expressing plasmid, atransfer vector plasmid, and an env-encoding plasmid. Four plasmidcotransfections were performed at a 3:3:3:1 molar ratio of a gag-polexpressing plasmid, a transfer vector plasmid, an env-encoding plasmid,and a rev-expressing plasmid. Five plasmid cotransfections wereperformed at a 3:3:3:1:1 molar ratio of a gag-pol expressing plasmid, atransfer vector plasmid, and an env-encoding plasmid, a rev-expressingplasmid and a vif-expressing plasmid. The culture supernatant containingviral vector particles was harvested 48 hours later, clarified with a0.45 μM membrane filter (Nalgene, NY, USA), and either used immediatelyor stored at −70° C. deep-freezer.

In Vitro Transduction

Transduction was carried out by adding the viral vector particles onto293T cells for 4 hours, in the presence of 8 μg/ml polybrene followed bythe addition of fresh media. After 48 hours Beta-Gal expression wasassayed after the cells were fixed in a solution consisting of 1%formaldehyde and 0.2% glutaraldehyde and stained for 12 hours at 37° C.in a solution containing 300 μg of 5-bromo-4-chloro-3-indolylyb-D-galactoside (X-Gal, Promega, WI, USA), 4 mM potassium ferrocyanide,4 mM potassium ferricyanide, and 2 mM Mgcl₂. Titers can be determined bycounting the number of blue foci as LacZ-forming units per ml (LFU/ml).

RT-PCR Assay

Total RNA was extracted from cultured cells or culture supernatant bythe method using TRIzol LS Reagent (GIBCO BRL, CA, USA). The total RNAwas treated with RNase free-DNase I (1 unit/μg of DNA for 20 minutes at37° C.) (Promega, WI, USA) to eliminate DNA contamination. The DNase Ireaction was stopped by adding RQ1 DNase stop solution provided withDNase I, and the RNA was cleaned up by the method using RNasy mini kit(Qiagen, Germany). The purified RNA was reverse transcribed into cDNA byreverse transcription (RT) reaction (90 min at 37° C.). In particular,the RT reaction was carried out in the presence of MuLV reversetranscriptase, oligo-dT primer or C-terminal specific primer, and dNTPsmix. PCR amplification was carried out for semi-quantitative analysis oftemplate DNA with specific primers. In particular, PCR product DNA wassynthesized from the cDNA or chromosomal DNA in the presence of heatstable Ex Taq polymerase, sequence specific DNA primers, and dNTPs mix.

Southern Blot Analysis

Genomic DNA was prepared from cells transduced with either pseudotypedHIV-1 or CAEV vector particles, and mock-transduced control cells usingthe DNeasy Tissue Kit (Qiagen, Germany). Ten μg of genomic DNA from theHIV-1 vector transduced cells were digested with BamH I and Kpn I. Tenμg each of the genomic DNA from the CAEV vector transduced cells and thenegative control cells were double digested with EcoR I and Ssp I. Thedigested genomic DNAs were separated by electrophoresis on 0.7% agarosegel and transferred onto positive charged nylon membrane (Roche,Germany). Dig-labeled probes were prepared by PCR with primers specificfor lacZ gene (Forward primer: CTGGCGTAATAGCGAAGAGG (SEQ ID NO: 65),Reverse primer: AACTCGCCGCACATCTGAAC (SEQ ID NO: 66)), and southernhybridization was carried out according to Dig application manual(Roche, Germany).

Growth Arrest of Cells and FACS Analysis of the Growth-Arrested Cells

293T cells were growth-arrested with aphidicolin (Sigma, USA)treatment(25 μg/ml), then transduced with CAEV viral vector particles.As a positive or negative control, cells were transduced side-by-sidewith either an HIV-1 vector or MuLV retrovirus vector. Two days aftertransduction, cells were stained with X-gal for beta-gal activity. Inthe aphidicolin treated culture, aphidicolin was present before andafter infection.

The growth arrest of cells was confirmed by FACS analysis. Theaphidicolin treated or untreated control cells were washed in PBS, fixedovernight in 70% ethanol at −20° C., and were followed by treatment ofpropidium iodide (100 μg/ml) (Sigma, USA) and RNAse A (100 μg/ml)(Qiagen, Germany) at RT for 1 hour. The cells were evaluated by FACSanalysis, and the percent of total viable cells in G1, S and G2/M phaseof the cell cycle was calculated (Becton Dickinson, Sanjose, Calif.).

EXAMPLE 1 Production of CAEV-Based Lentiviral Vector Particles

Replication defective lentiviral vector particles were generated bytransient co-transfection of human 293T cells with a minimum ofthree-plasmid system of a CAEV gag-pol expressing plasmid, a CAEVenv-expressing plasmid and a transfer vector plasmid. In a four-plasmidsystem, a CAEV rev expressing plasmid is added, and in a five-plasmidsystem, a CAEV vif expressing plasmid is added. For efficient packaging,transfer vectors were designed to contain the beginning of thegag-encoding sequence, where mutations were introduced into the startATG codon and an ATG codon located downstream (ATG to TAG) to preventthe expression of gag proteins. RRE was included to boost packagingefficiency and the rev in the four- and five-plasmid systems wasexpressed from the vector to support the CAEV mRNA export. The internalHCMV-MIEP promoter-driven β-galactosidase gene in the transfer vectorplasmid was inserted to serve as a reporter gene. The U3 region of the5′LTR was replaced with the strong viral promoter, HCMV-MIEP, allowingthe vector genome to be tat independent.

Transfer Vector RNA Transcription Level.

Transcription level of genomic RNA from a transfer vector is one of thecritical factors mediating high titer production of recombinant viralvectors from packaging cells. In the present invention, HCMVenhancer/promoter element was used to construct the HCMV/CAEV hybrid LTRpromoter system for safe and efficient transcription of the transfervector RNA. To examine the transcription level of the transfer vectorplasmids of the pCAH/SINd (SEQ ID NOs: 67-71, 73, 78, and 79) seriescontaining the hybrid LTR promoter, each of the transfer vector plasmidswas introduced into human T cells, together with the packaging plasmids(pMGP/RRE (SEQ ID NO: 77), pHYK/rev (SEQ ID NO: 75), pHYK/vif (SEQ IDNO: 76), pHGVSV-G (SEQ ID NO: 74) or pMYKEF1/env (SEQ ID NO: 72)), byliposome-mediated transfection. After 48 hours of incubation, total RNAwas purified from the transfected cells and was subjected to ReverseTranscriptase Polymerase Chain Reaction (RT-PCR) analysis for the vectorRNA transcript measurement. The PCR primer set (RRE primer set) for theCAEV transfer vectors was designed for synthesizing 348-bp PCR productcoding for a part of RRE region. Another PCR primer set (lacZ primerset) for the HIV-1 transfer vector, pHRlacZ (Naldini et al., 1996), wasdesigned for synthesizing the 645 bp PCR product coding for a part ofthe lacZ gene. As shown in FIG. 7, the CAEV transfer vectors of thepresent invention produced RNA transcript at a level comparable to thatof the HIV- 1-based lentiviral transfer vector.

Formation and Release of the Vector Particles.

To examine the formation and release of mature and infectious virusvector particles, CAEV vector particles were produced followingliposome-mediated co-transfection of the pMGP/RRE (SEQ ID NO: 77)gag-pol expression plasmid, the pHGVSV-G (SEQ ID NO: 74) env expressionplasmid, the pHYK/rev (SEQ ID NO: 75) rev expression plasmid, pHYK/vif(SEQ ID NO: 76) vif expression plasmid, and the pCAH/SINd60/hlacZ (SEQID NO: 78) transfer vector plasmid into human 293T cells (DuBridge etal., 1987). Forty eights hours after transfection, the culturesupernatant was harvested from the transfected cells and applied tofresh human 293T cells in the presence of 8 μg/ml polybrene forinfection. The results indicated that the five plasmids system of thepresent invention was capable of producing comparable viral vectorparticle titers to that of the MuLV-based retroviral vector system(pEQPAM3, pMFG/lacZ/puro, pHGVSV-G (SEQ ID NO: 74)) (Ory, Neugeboren,and Mulligan, 1996; Persons et al., 1998) (shown in FIG. 8).

EXAMPLE 2 Effect of Rev and Vif Expression on Vector Particle Production

To determine the effect of CAEV rev and vif regulatory gene expressionon vector particle production, the vector particle production system of(1) the three-plasmid system (pCAH/SIN, pMGP/RRE (SEQ ID NO: 77),pHGVSV-G (SEQ ID NO: 74) or pMYKEF1/env (SEQ ID NO: 72)), which isdevoid of rev- and vif-encoding sequences, (2) the four-plasmids system(pCAH/SIN, pMGP/RRE (SEQ ID NO: 77), pHGVSV-G (SEQ ID NO: 74) orpMYKEF1/env (SEQ ID NO: 72), pHYK/rev (SEQ ID NO: 75)), which is devoidof vif-encoding sequence and (3) the five-plasmid system (pCAH/SIN,pMGP/RRE (SEQ ID NO: 77), pHGVSV-G (SEQ ID NO: 74) or pMYKEF1/env (SEQID NO: 72), pHYK/rev (SEQ ID NO: 75), pHYK/vif (SEQ ID NO: 76)), whichcontains both rev- and vif-encoding sequences were tested side by sidefor their efficiency in vector particle production. The plasmids of eachsystem were transfected into 293T cells. At day 2 post-transfection, thetransfer vector RNA and the virion RNA were extracted from thetransfected cells and the culture medium of the transfected cells,respectively, and used as RT-PCR templates with the lacZ primer set todetect the transfer vector RNA genome.

As shown in FIG. 9, although the expression level of the transfer vectorRNA in the packaging cells was independent of the expression of the revor the vif genes (Lane 1, 2 and 3 in FIG. 9), the amount of theencapsidated transfer vector RNA in the absence of rev (Lane 4, FIG. 9)was much lower than that in the presence of rev (Lane 5 in FIG. 9).Surprisingly, however, the titer of the vector particles measured byRT-PCR with the encapsidated RNA in the presence of vif (Lane 6 in FIG.9) was lower than the vector particles measured by the RT-PCR with theencapsidated RNA in the absence of CAEV vif (Lane 5 in FIG. 9). Thesedata indicate that CAEV rev and vif are not required for vector particleproduction, but rev is preferred for efficient vector particleproduction.

Of note is that the results of the present invention regarding the vifexpression is inconsistent with the observations reported by Harmache etal. (Harmache et al., 1995; Harmache et al., 1996), where the vif genewas reported to be essential for efficient replication of CAEV in thegoat synovial membrane cells and to be affecting the late steps of thevirus replication cycle (e.g., RNA encapsidation, release of virusparticles from host cells). One plausible explanation for theinconsistency may be in the use of the human 293T cells instead of thegoat cells in the production of the recombinant CAEV vector particles.This interpretation supports the hypothesis proposed by Seroude et al.that the species-specific restrictions between vif and thevirus-producing cells may modulate the vif function on viral infectivity(Seroude et al., 2002).

EXAMPLE 3 Identification of the Optimal Packaging Signal Sequence

To identify the optimal packaging signal sequence for the encapsidationof CAEV transfer vector RNA, a series of plasmids containing differentportions of the CAEV gag-coding region and the untranslated regionbetween the 5′LTR and the gag start codon were compared for their vectorparticle production efficiency as follows. Human 293T cells wereco-transfected with the pMGP/RRE (SEQ ID NO: 77) gag-pol expressionplasmid, the pHGVSV-G (SEQ ID NO: 74) env expression plasmid, thepHYK/rev (SEQ ID NO: 75) rev expression plasmid, the pHYK/vif (SEQ IDNO: 76) vif expression plasmid, and the pCAH/SINd (SEQ ID NOs: 67-71,73, 78, and 79) transfer vector series plasmid. As a negative control, aCAEV transfer vector pCAM/lacZ(L) was transfected in the absence ofpackaging plasmids. On day 2 post-transfection, virion RNA was extractedfrom the culture medium of the transfected cells and used as an RT-PCRtemplate with the RRE primer set to detect the CAEV transfer vectorseries RNA genome, or with the lacZ primer set to detect the HIV-1transfer vector RNA genome. As shown in FIG. 10, a strong PCR productsignal, indicating efficient release of the virus particles containingthe viral RNA, was obtained from the culture medium harvested from thevirus producing 293T cells transfected with pCAH/SINd1 (SEQ ID NO: 68),which contained the complete 5′LTR as well as the first 327 bp of thegag region (lane 3 in FIG. 10). This signal was comparable to thatobtained with the positive control, the HIV-1 vector, indicating thatthe amount of the encapsidated CAEV transfer vector RNA of the presentinvention is comparable to that of the HIV-1-based transfer vectors(lane 8 in FIG. 10). The packaging efficiency of the CAEV transfervectors with gag-coding region of the first 612 bp or longer wassignificantly reduced (lanes 4, 5, and 6). The PCR product signals werenot detectable when the transfer vectors used were devoid of thegag-coding sequences (lane 1 and 2 in FIG. 10). Negative control wastransfected with a transfer vector only, and the positive control, HIV-1vector, was transfected with pCMVΔR8-2, pHR′/lacZ and pHGVSV-G (SEQ IDNO: 74) (lanes 7 and 8 in FIG. 10).

In conclusion, the transfer vector RNAs were encapsidated efficiently inthe packaging cells only when the transfer vectors included less thanabout 600 bp of the N-terminal gag-coding sequences as well as theentire untranslated region between the 5′LTR and the gag start codon.These results indicate that the role of the secondary structure of theRNA within the packaging signal is more important than the primarystructure in RNA encapsidation.

EXAMPLE 4 Pseudotyping of the CAEV Vector Virion

To determine whether the recombinant CAEV vector virion can bepseudotyped with the GaLV glycoprotein as well as the VSV-Gglycoprotein, either the GaLV expression vector, pMYKEF1/env (SEQ ID NO:72), or the VSV-G expression vector, pHGVSV-G (SEQ ID NO: 74), wascotransfected with a transfer vector plasmid and the packaging plasmidsinto human 293T cells. Forty eights hours after transfection, culturesupernatant containing pseudotyped virion particles released from thetransfected cells was harvested, clarified with a 0.45 μm membranefilter, and used for infecting 293T human target cells. One day afterinfection, genomic DNA was purified by using a Genomic DNA Isolation kit(Qiagen, HL, Germany) and subjected to PCR experimentation to detect theintegrated proviral cDNA. As expected, CAEV vector (Lane 1 in FIG. 11)was pseudotyped efficiently with the VSV-G protein, comparable to theMuLV- (Lane 3 in FIG. 11) and the HIV-1-based vector (Lane 4 in FIG.11). In addition, inconsistent to the HIV-1 lentiviral vector system,the CAEV vector of the present invention was pseudotyped successfullywith the GaLV envelope (Lane 2 in FIG. 11). This pseudotyping ability ofthe CAEV vectors with the GaLV envelope can afford a great advantage inthe development of a clinical grade lentiviral vector system. MuLV(transfected with pEQPAM3, pMFG/lacZ/puro and pHGVSV-G (SEQ ID NO: 74))and HIV-1 (transfected with pCMVΔR8-2, pHR′/lacZ and pHGVSV-G (SEQ IDNO: 74)) vector controls in lanes 3 and 4, respectively.

EXAMPLE 5 Generation of a CAEV Packaging Cell Line

Both the pMGP/RRE (SEQ ID NO: 77) and the pHYK/rev (SEQ ID NO: 75)vectors encode a neo^(r) gene for selection in eukaryotic cells. Forefficient selection after cotransfection with a gag-pol and a revexpression vectors, another CAEV gag-pol expression vector may beconstructed by replacing the neo^(r) gene with the other antibioticresistance genes such as bacterial gpt gene, or one packaging plasmidsystem encoding the gag, pol and rev genes can be used. To determine ifstable 293T cells expressing CAEV packaging proteins could be generated,antibiotic resistant colonies are selected under selective medium.Production of recombinant CAEV vector from the stable 293T cellssuggests the feasibility of generating stable packaging cell lines forCAEV vector production.

EXAMPLE 6 Integration of the CAEV-based Vector cDNA into the HostChromosome

To examine the integration of the CAEV vector cDNA after transduction,the CAEV vector particles were produced by liposome-mediatedco-transfection of the pMGP/REV/RRE gag-pol expression plasmid, thepHGVSV-G (SEQ ID NO: 74) env expression plasmid, and thepCAH/SINd1/hlacZ (SEQ ID NO: 79) transfer vector plasmid into human 293Tcells. As a positive control, the pCMVΔR8.2 gag-pol expression plasmid,the pHGVSV-G (SEQ ID NO: 74) env expression plasmid, and the pHR/lacZtransfer vector were co-transfected into the 293T cells to produce theHIV-1 vector particles. As a negative control, only the pCAH/SINd1/hlacZ(SEQ ID NO: 79) transfer vector plasmid was transfected. Forty eighthours after transfection, the culture supernatants were harvested fromeach of the transfected cells and applied to fresh 293T cells in thepresence of 8 μg/ml polybrene for infection. After 48 hours, genomic DNAwas prepared from each of the transduced cells, followed by southernblot assay after restriction enzyme digestion. The Dig-labeled lacZprobes detected 3.15 kb BamH I-Kpn I fragment for the HIV-1-basedtransfer vector, and 1.35 kb Hind III-Ssp I fragment for the CAEV-basedtransfer vector and the negative control. For the positive controls, the0.3 ng and 3 ng of Hind III-Ssp I DNA fragment of the pCAH/SINd1/hlacZ(SEQ ID NO: 79) transfer vector plasmid were used. As shown in FIG. 12,the CAEV-based transfer vector of the present invention was integratedat a level comparable to that of the HIV-1-based lentiviral transfervector.

EXAMPLE 7 Gene Transfer to Non-dividing Cells

293T cells were treated with the DNA synthesis inhibitor, aphidicolin,plated on a 6-well Culture plate, and then transduced with the CAEVvector particles encoding a lacZ marker gene. As controls, cells wereinfected side-by-side with a lacZ expressing MuLV retroviral vector andHIV-1 lentiviral vector. At 48 hours after infection, in order toexamine the trasduction efficiency, expression of the transduced lacZgene was counted by X-gal staining. As shown in FIG. 14, theMuLV-derived vector efficiently infected cells not treated with the DNAsynthesis inhibitor. However, when cells were arrested in the cell cycleby the DNA synthesis inhibitor treatment, the transduction efficiencywas dropped markedly. In contrast, the CAEV-based vector was capable ofefficiently transducing non-dividing human cells as well as dividingcells at a level comparable to that of the HIV-1-based vector.

EXAMPLE 8 In Vivo Transduction of Muscle Cells

In this example, the CAH/SINd1/hlacZ (SEQ ID NO: 79) CAEV vector is usedto transduce muscle cells in vivo. The hind-legs of mice (Beige strain)are intramuscularly injected with 100 μl of the CAEV vectors in thepresence of 4 μg/ml of polybrene. The mice are sacrificed two days laterand the injected tissue is prepared for frozen section and forβ-galactosidase analysis. The expected result is that CAH/SINd1lacZ (SEQID NO: 79) CAEV vector transduces muscle cells efficiently in vivo.

The foregoing specification, including the specific embodiments andexamples, is intended to be illustrative of the invention and is not tobe taken as limiting. Numerous other variations and modifications can beeffected without departing from the true spirit and scope of theinvention. All publications, including sequences deposited in the NCBIdatabase, patents and patent applications cited herein are incorporatedby reference in their entirety into the disclosure.

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Name: NC_001463 (SEQ ID NO: 1) Len: 9300Check: 3957 Weight: 0 Name:  AF322109 (SEQ ID NO: 2) Len: 9300 Check:5441 Weight: 0 // 1                                                   50NC_001463 .......... ....GAGTTC TAGG...AGA GTCCCTCCTA GTCTCTCCTC AF322109 GTGAGTGCTC TGAGGAGCTC GAAGGAAAGA GTCC.TC..A GCCTCTCCTC51                                                 100 NC_001463TCCGAGGAGG TACCGAGACC TCAAAATAAA GGAGTGATTG CCTTACTGCC  AF322109TCCGAGGAGC TTCGG....C TCATAATAAA GGAGTGCTTG CTTCA..ACA101                                                150 NC_001463GAGTGGAGAG TGATTACTGA GCGGCCGGTG TATCGGGAGT CGTCCCTTAA  AF322109GAACTGAG.. ......CTGG TCGTGGTTAT TATCGGG... .GACCGAAGT151                                                200 NC_001463TCTGTGCAAT ACCAGAGCGG CTCTCGCAGC TGGCGCCCAA CGTGGGGCCC  AF322109CCCGTGCAAC ACCGGGGCGG TTCTCGCAGC TGGCGCCCAA CGTGGGGCTC201                                                250 NC_001463GAGGAG.... .......... .......... .......... ..........  AF322109GAGTAGCTTG AGAAGCTCGA CTGAGATCTG AATCCAAGAG CGACATCAGA251                                                300 NC_001463....AAGAAA AGAAAGC... GGCCCTGAGA ACTCGGCTTC TG..AAAAAG  AF322109CAGCAAGAAA TGAGAGTAAT GAGACCGCGA GCTCTGCTGC TGTAAAAAAG301                                                350 NC_001463AGGAAGAGGA CAAGTTGCTA TAGCAACAAG AGAGAAGAAG TAGAGCAAAG  AF322109AGGAAGTAG. CGGGTTGCCG AGGCAACTGC TCAGAAGAAC CAGGGGAAAG351                                                400 NC_001463GTCCAGTGGC T.CGGAAAAA GAGGAACTGA AACTTCGGGG ACGCCTGAAG  AF322109GGCTTCCAGC AACCTCAAAA GAGGAACCGA GACTTCGGGG ACGCCTGAA.401                                                450 NC_001463GAGTAAGGTA AGTGACTCTG CTGTACGCGG GGCGAGGCAG AGGTT.TCCT  AF322109..GTAAGGTA AGTGACTCTG CTGTACGCGG GGCGAGGCAT AGGAGATCCT451                                                500 NC_001463TCTAAATT.G AAAGAGAAGT GTTGCTGCGA GAGGTCTTGG TGGTCGAGAA  AF322109TCTATTCTAG GAAGAGAAGC GCTGTTCTGG GAGGTCTTGG CGACCGAGAA501                                                550 NC_001463TCCTGTACAA AAAAAAGGAG GGATCTCGGT CAGGACCAGG ACCCCTGGGA  AF322109TCTTGTT... AAATAAGCCA GGATCTCGAT CAGGACCAAG ACCCCTCAGG551                                                600 NC_001463GTAATACAAC AGCAACACCG TAAGAAAATC CGCCATGGTG AGTCTAGATA  AF322109AGAGGGTATA GACAGCGTGG TAAGAAA.TC CGCCGTGGTG AGTCTAGATA601                                                650 NC_001463GAGACATGGC GAGGCAAGTC TCCGGGGGGA AAAGAGATTA TCCTGAGCTC  AF322109GAGACATGGT GAGGCAGGCC TCCGGAAGGG GAAAGGAGTA CCCCGAGCTA651                                                700 NC_001463GAAAAATGTA TCAAGCATGC ATGCAAGATA AAAGTTCGAC TCAGAGGGGA  AF322109AAAGAATGTC TGAAAAAGGC ATGCAAAATA AAAGTAAGGG CTGGGGGGGA701                                                750 NC_001463GCACTTGACA GAAGGAAATT GTTTATGGTG CCTTAAAACA TTAGATTACA  AF322109GCGCCTGACA GAAGGAAATT GTCTCTGGTG TATAAAAACA CTAGAGTGTA751                                                800 NC_001463TGTTTGAGGA CCATAAAGAG GAACCTTGGA CAAAAGTAAA ATTTAGGACA  AF322109TGTATGAGGA TTGTAGGGAG GAACCTTGGA CCCCAGAAAA ATGTAAACAA801                                                850 NC_001463ATATGGCAGA AGGTGAAGAA TCTAACTCCT GAGGAGAGTA ACAAAAAAGA  AF322109TTATGGAAAA AGTTGAAGCA GGTAGAGCCT GAGGAGAGTA GCAAAGCAGA851                                                900 NC_001463CTTTATGTCT TTGCAGGCCA CATTAGCGGG TCTAATGTGT TGCCAAATGG  AF322109CTATAACTCG TTAAAAGCAA CCTTGGCGGG GATAGTCTGT GTGCAAATGG901                                                950 NC_001463GGATGAGACC TGAGACATTG CAAGATGCAA TGGCTACAGT AATCATGAAA  AF322109GAATGCAGCC CGAGACACTG CAGGATGCGA TAGCAACCTT AAACATGAGA951                                               1000 NC_001463GATGGGTTAC TGGAACAAGA GGAAAAGAAG GAAGACAAAA GAGAAAAGGA  AF322109GA........ TGAAGTAAAA GGAAAGGAA. .AAGCCATCA GAAGAAAAGA1001                                              1050 NC_001463AGAGAGTGTC TTCCCAATAG TAGTGCAAGC AGCAGGAGGG AGAAGCTGGA  AF322109AGGGAATATA TCCC..ATAT TAGTGCAGGC AGGAGGAGGA AGAGCATGGA1051                                              1100 NC_001463AAGCAGTAGA TTCTGTAATG TTCCAGCAAC TGCAAACAGT AGCAATGCAG  AF322109GAGCGGTAGA GCCTGCTACC TTTCAGCAGC TCCAAACAGT GGCAATGCAG1101                                              1150 NC_001463CATGGCCTCG TGTCTGAGGA CTTTGAAAGG CAGTTGGCAT ATTATGCTAC  AF322109CATGGACTAG TATCAGAAGA ATTTGAAAGG CAGCTAGCAT ACTATGCCAC1151                                              1200 NC_001463TACCTGGACA AGTAAAGACA TACTAGAAGT ATTGGCCATG ATGCCTGGAA  AF322109CACATGGACA AGCAAGGATA TCTTAGAAGT ATTAGCCATG ATGCCAGGAA1201                                              1250 NC_001463ATAGAGCTCA AAAGGAGTTA ATTCAAGGGA AATTAAATGA AGAAGCAGAA  AF322109ATAGAGCGCA AAAAGAACTA ATACAAGGAA AGTTAAATGA GGAAGCAGAG1251                                              1300 NC_001463AGGTGGAGAA GGAATAATCC ACCACCTCCA GCAGGAGGAG GATTAACAGT  AF322109AGATGGAGAA GGCAGAATCC ACAACCT... ...GCGGGCG GGTTAACCGT1301                                              1350 NC_001463GGATCAAATT ATGGGGGTAG GACAAACAAA TCAAGCAGCA GCACAAGCTA  AF322109GGATCAGATA ATGGGGGTAG GACAAACGAA TCAGGCAGCG GCACAGGCTA1351                                              1400 NC_001463ACATGGATCA GGCAAGGCAA ATATGCCTGC AATGGGTAAT AAATGCATTA  AF322109ATATGGATCA AGCAAGACAA ATATGCCTAC AATGGGTTAT AACAGCAATA1401                                              1450 NC_001463AGAGCAGTAA GACATATGGC GCACAGGCCA GGGAATCCAA TGCTAGTAAA  AF322109AGAGGAGTTA GGCATATGGC CCATAGACCA GGAAATCCCA TGCTGGTAAG1451                                              1500 NC_001463GCAAAAAACG AATGAGCCAT ATGAAGATTT TGCAGCAAGA CTGCTAGAAG  AF322109ACAAAAACCA AATGAGAACT ATGAAGAGTT TGCCGCAAGG TTGTTAGAAG1501                                              1550 NC_001463CAATAGATGC AGAGCCAGTT ACACAGCCTA TAAAAGATTA TCTAAAGCTA  AF322109CAGTGGATGC AGAACCCGTT ACCCAACCTA TAAAAGAATA TTTAAAGGTA1551                                              1600 NC_001463ACACTATCTT ATACAAATGC ATCAGCAGAT TGTCAGAAGC AAATGGATAG  AF322109ACTCTGTCTT ACACAAATGC AAATTCGGAA TGTCAAAAAC ATATGGACAG1601                                              1650 NC_001463AACACTAGGA CAAAGAGTAC AACAAGCTAG TGTAGAAGAA AAAATGCAAG  AF322109AGTGTTGGGG CAAAGAGTAC AGCAGGCCTC AATAGAAGAA AAAATGCAGG1651                                              1700 NC_001463CATGTAGAGA TGTGGGATCA GAAGGGTTCA AAATGCAATT GTTAGCACAA  AF322109CATGCAGGGA CATCGGGGGA ACAGCATATC AGATGCAGTT GCTTGCACAA1701                                              1750 NC_001463GCATTAAGGC CAGGAAAAGG AAAAGGGAAT GGACAGCCAC AAAGGTGTTA  AF322109GCCCTCCGTG GCGGAAAAGA AGATGGGAAA AAATCTGTAG GGAAGTGTTA1751                                              1800 NC_001463CAACTGTGGA AAACCGGGAC ATCAAGCAAG GCAATGTAGA CAAGGAATCA  AF322109TAACTGTGGA AGGCCCGGAC ACAGAGCAAA AGAATGCAGA CAAGGCATTA1801                                              1850 NC_001463TATGTCACAA CTGTGGAAAG AGAGGACATA TGCAAAAAGA ATGCAGAGGA  AF322109TATGTCACAA CTGTGGAAAA AGAGGGCATA TACAGAAAAA CTGCA....A1851                                              1900 NC_001463AAGAGAGACA TAAGGGGAAA ACAGCAGGGA AACGGGAGGA GGGGGATACG  AF322109AC.AGAA... .AAGAAGAAA GGAGCAGGGA AACATGAGGA GGGGGCTACG1901                                              1950 NC_001463TGTGGTGCCG TCCGCTCCTC CTATGGAATA ACTTCAGCAC CACCTATGGT  AF322109TGTGGTGCCG TCCGCACCCC CTATGGAGTA ACGCAAGCAC CACTAATAGT1951                                              2000 NC_001463TCAGGTCCGC ATAGGTTCCC AGCAGAGGAA CTTGTTATTT GATACCGGGG  AF322109TAGGGTACAA ATAGGGAATC AGGAGAAACA ATTATTATTT GACACAGGGG2001                                              2050 NC_001463CGGACCGAAC TATAGTTAGA TGGCATGAGG GCTCGGGAAA CCCAGCCGGA  AF322109CAGATAAAAC GATAGTAAGA ATGCATGATG GAACAGGGAT TCCAAACGGA2051                                              2100 NC_001463AGGATAAAAC TGCAAGGAAT AGGAGGAATA GTAGAAGGAG AAAAATGGAA  AF322109AGAATAAAAT TACAAGGGAT AGGAGGAATA GTAGAAGGAG AAAAATGGAA2101                                              2150 NC_001463TAATGTAGAA TTAGAATATA AAGGAGAAAC AAGAAAGGGA ACAATAGTAG  AF322109TAAAGTACCC ATGACATATA AGGGAGAAAC ATCCTGCCCA AGCTTGGTTG2151                                              2200 NC_001463TGTTACCACA AAGTCCAGTA GAAGTATTAG GACGAGATAA CATGGCCCGA  AF322109TGCTAAGAGA TAGCCCAGTA GAAGTATTGG GAAGAGATAA CATGGAAGCA2201                                              2250 NC_001463TTTGGAATAA AGATAATAAT GGCAAATTTA GAGGAAAAAA GAATCCCAAT  AF322109TTCGGCGTAA CCCTAATAAT GGCAAATTTA GAAGATAAGA AAATTCCCAC2251                                              2300 NC_001463TACAAAAGTA AAATTGAAAG AGGGATGTAC GGGTCCACAT GTCCCACAAT  AF322109AATACCAGTA GAATTGAAAG AAGGATGTAA AGGGCCACAT GTGCCCCAGT2302                                              2350 NC_001463GGCCATTAAC AGAAGAGAAA TTAAAAGGTC TAACAGAAAT CATAGATAAA  AF322109GGCCATTAAC AGCAGAGAAA TTACAAGGAC TAACAGGAAT AGTAGAAAAA2352                                              2400 NC_001463TTAGTGGAAG AAGGAAAACT AGGAAAGGCA CCCCCACATT GGACATGTAA  AF322109TTACTACAGG AAGGAAAATT GGCAGAGGCC CCAGAGGGAT GGACGTGGAA2402                                              2450 NC_001463TACTCCAATC TTTTGCATAA AAAAGAAATC AGGGAAGTGG AGAATGTTAA  AF322109CACGCCCATC TTCTGCATAA AAAAGAAGTC AGGAAAATGG AGAATGTTAA2451                                              2500 NC_001463TAGATTTCAG AGAATTGAAC AAACAGACAG AAGATTTAAC AGAAGCGCAG  AF322109TAGATTTTAG GGAATTAAAT AAGCAAACAG CAGATTTAGC AGAAGCGCAG2502                                              2550 NC_001463TTAGGACTCC CGCATCCGGG AGGACTACAA AAGAAAAAAC ATGTTACAAT  AF322109CTAGGACTGC CACACCCAGG AGGGTTGCAA AGGAAAAAGA ATGTAACAAT2551                                              2600 NC_001463ATTGGACATA GGAGATGCAT ATTTTACTAT ACCCCTATAT GAACCATATC  AF322109TCTGGACATA GGAGATGCAT ATTTCACAAT TCCCTTATAC GAGCCCTATC2601                                              2650 NC_001463GAGAGTACAC ATGTTTTACT CTATTAAGTC CTAATAATCT AGGACCATGT  AF322109AGAAATATAC ATGCTTCACA CTCCTAAGTC CTAACAATTT GGGACCATGT2651                                              2700 NC_001463AAAAGATACT ATTGGAAAGT GCTGCCACAA GGTTGGAAAT TGAGTCCATC  AF322109AAAAGGTATT ATTGGAAAGT ATTACCCCAG GGATGGAAAT TGAGCCCAGC2701                                              2750 NC_001463TGTATATCAA TTTACTATGC AGGAGATCTT AGAGGATTGG ATACAGCAGC  AF322109TGTATATCAA TTCACCATGC AAAGGTTGTT AAAAGGATGG ATACAACAGC2751                                              2800 NC_001463ATCCAGAAAT TCAATTTGGC ATATATATGG ATGATATTTA CATAGGAAGT  AF322109ATAAAAACAT ACAATTTGGA ATATATATGG ATGATATCTA TATTGGAAGT2801                                              2850 NC_001463GATTTAGAAA TTAAAAAGCA TAGAGAAATA GTGAAAGATT TAGCCAATTA  AF322109GATCTAACGA TAGCCCAACA TAGGAAGATA ATAGAAGAAT TAGCCTCATT2851                                              2900 NC_001463TATTGCCCAA TATGGATTCA CTCTGCCAGA AGAGAAGAGA CAAAAGGGAT  AF322109TATAGAACAA TTTGGGTTTA CATTACCAGA AGATAAGAGA CAAGAGGGCT2901                                              2950 NC_001463ATCCAGCAAA ATGGCTAGGA TTTGAACTAC ACCCGCAGAC CTGGAAATTT  AF322109ATCCAGCAAA ATGGCTAGGA TTCGAGCTAC ATCCAGAAAA ATGGAAATAT2951                                              3000 NC_001463CAGAAGCATA CATTACCTGA ATTAACAAAG GGAACAATAA CATTAAATAA  AF322109CAAAAGCATA AATTGCCGGA ATTACAAGAG GGGGTAATAA CCCTGAACAA3001                                              3050 NC_001463ATTACAGAAA TTAGTAGGAG AATTAGTATG GAGACAATCC ATAATTGGGA  AF322109ATTACAGAAG ATAGTAGGGG AATTAGTGTG GAGACAATCC TTGATAGGAA3051                                              3100 NC_001463AAAGCATTCC TAACATTCTG AAATTAATGG AAGGAGATAG AGAATTACAA  AF322109AGAGCATCCC CAATATCATA AAATTAATGG AAGGAGATCG CGCATTACAA3101                                              3150 NC_001463AGTGAAAGAA AAATTGAAGA AGTACATGTG AAAGAATGGG AAGCATGTAG  AF322109AGTGAAAGGA AAATAGAAAG AATACATGTA CAAGAATGGG AAGCATGTCA3151                                              3200 NC_001463GAAAAAATTA GAAGAAATGG AAGGAAATTA TTATAATAAA GACAAAGATG  AF322109AAAGAAATTA GATGAAATGG TAGGAAATTA TTACAGAGAA GAAGAAGATA3201                                              3250 NC_001463TCTATGGACA ATTGGCTTGG GGAGACAAAG CTATAGAATA TATAGTGTAT  AF322109TCTATGGACA AATAACTTGG GGGGATAAGG CAATAAAATA CATAGTATTC3251                                              3300 NC_001463CAGGAGAAAG GGAAACCATT ATGGGTAAAT GTGGTTCACA ATATAAAGAA  AF322109CAAAGGAAAG GGGAACCCCT ATGGGTAAAT GTAGTACATG ACATAAAAAA3301                                              3350 NC_001463CCTAAGCATC CCGCAACAGG TTATTAAAGC AGCGCAAAAA TTAACCCAAG  AF322109TTTGAGTCTC CCACAGCAAG TGATAAAAGC AGCACAGAAA TTAACCCAGG3351                                              3400 NC_001463AAGTCATCAT TAGGACAGGA AAAATACCAT GGATATTGTT GCCAGGGAAA  AF322109AAGTAATCAT AAGAACAGGA AAAATCCCAT GGCTGCTACT ACCAGGAAGA3401                                              3450 NC_001463GAAGAAGATT GGAGACTAGA ATTGCAATTA GGGAACATCA CATGGATGCC  AF322109GAAGAAGACT GGAGATTAGA ACTGCAGGTA GGGAACATCA CGTGGATGCC3451                                              3500 NC_001463AAAATTTTGG TCCTGTTATC GAGGA.CATA CAAGATGGAG AAAAAGAAAT  AF322109ATCATTTTGG TCATGTTATC GAGGAGCACC CAAG.TGGAA AAGAAGGAAC3501                                              3550 NC_001463ATAATAGAAG AAGTAGTAGA AGGGCCTACA TATTATACAG ATGGAGGAAA  AF322109ATAGTGGCAG CAGTGGTAGA TGGACCGACA TATTATACAG ATGGGGGAAA3551                                              3600 NC_001463AAAGAATAAA GTAGGAAGTC TAGGGTTCAT AGTATCAACA GGGGAAAAAT  AF322109GAAAAACGCA CAGGGAAGCT TTGGCTTCAT CTCCCCAACA GGAGAAAAGT3601                                              3650 NC_001463TTAGAAAGCA TGAAGAGGGC ACAAACCAGC AACTAGAATT AAGAGCCATA  AF322109TCAGAAGGCA TGAAGATGGA ACTAATCAGG TATTAGAATT AAGGGCAATA3651                                              3700 NC_001463GAGGAAGCTC TAAAACAAGG GCCTCAAACA ATGAATTTAG TAACAGATAG  AF322109GAAGATCCAT GTAAACAAGG ACCTGAAAGC ATGAACATTG TAACTGACAG3701                                              3750 NC_001463TAGATATGCA TTTGAATTTT TATTAAGAAA TTGGGATGAA GAAGTAATAA  AF322109CAGGTATGCT TATGAATTCA TGCTCCGAAA CTGGGATGAA CAGGTCATAA3751                                              3800 NC_001463AGAATCCAAT TCAAGCAAGA ATTATGGAAA TTGCCCACAA GAAAGATAGG  AF322109GAAACCCCAT TCAGGCAAGA ATCATGGCAG AAGTGCACAA GAAAAAGCAG3801                                              3850 NC_001463ATAGGAGTGC ATTGGGTGCC AGGACATAAA GGGATTCCCC AAAATGAAGA  AF322109GTAGGAATAC ACTGGGTGCC AGGGCATAAA GGAATACCTC AGAATGAAGA3851                                              3900 NC_001463AATAGACAAA TATATTTCGG AAATATTTCT TGCAAAAGAA GGAGAAGGAA  AF322109GATAGACCAG TACATATCAG AAGTATTCTT AGCACGAGAA GGAACAGGGA3901                                              3950 NC_001463TTCTCCCAAA AAGAGAAGAG GATGCAGGGT ATGATTTAAT ATGCCCAGAA  AF322109TATGTGAAAA AAGGAAGGAA GATGCTGGAT ATGATTTATT ATGCCCGCAT3951                                              4000 NC_001463GAGGTTACCA TAGAGCCAGG ACAAGTGAAA TGCATCCCCA TAGAGCTAAG  AF322109GAGGTAATAC TTAAACCCCA AGAAGTAAAA CGGATCCCAA TAGACCTAAA4001                                              4050 NC_001463ATTAAATTTA AAGAAATCAC AATGGGCTAT GATTGCTACA AAAAGCAGCA  AF322109ATTAAAATTG AAAGAAAAGC AATGGGCCAT GATAAGTGGG AAAAGTAGCG4051                                              4100 NC_001463TGGCTGCCAA AGGAGTGTTC ACACAAGGAG GAATCATAGA CTCAGGATAT  AF322109TTGCAGCAAA AGGAATATTT GTACAAGGAG GCATAATAGA TTCAGGGTAT4101                                              4150 NC_001463CAGGGACAAA TACAGGTAAT AATGTATAAT AGCAATAAAA TAGCAGTAGT  AF322109CAGGGACAAG TACAAGTCAT CCTATATAAT AGTAATAAGA TAGAGGTCAA4151                                              4200 NC_001463CATACCCCAA GGGAGAAAAT TTGCACAATT AATATTAATG GATAAAAAGC  AF322109AATACCACAA GGCAGGAAAT TTGCCCAATT AATATTAATG AACTTACAAC4201                                              4250 NC_001463ATGGAAAATT GGAACCCTGG GGGGAAAGCA GAAAAACAGA AAGGGGAGAA  AF322109ATGAAGAATT AGAAGAATGG GGAAAGGAAA GAAAAACAGA AAGAGGAACA4251                                              4300 NC_001463AAAGGATTTG GGTCTACAGG AATGTATTGG ATAGAAAATA TTCCTCTGGC  AF322109AAAGGATTTG GGTCTACAGG AGCATTTTGG ATAGAGAATA TTCCCCAAGC4301                                              4350 NC_001463AGAGGAAGAC CACACAAAAT GGCATCAAGA TGCCCGATCA TTGCATCTAG  AF322109AGAGGAAGAA CATTACAAAT GGCATCAAGA TGCTAGATCT CTGCAGCTAG4351                                              4400 NC_001463AATTTGAAAT TCCAAGAACA GCAGCAGAAG ACATAGTAAA TCAATGTGAA  AF322109AATTCAAGAT ACCTAGAGCA GCAGCAGAAG ACATTATACA GCACTGTGAG4401                                              4450 NC_001463ATATGCAAAG AAGCGAGGAC ACCTGCAGTA ATTAGAGGCG GAAACAAAAG  AF322109GTATGTCAAG AAGGCAAACC CGCAGCGATC ACGAGAGGGG GAAATAAAAG4451                                              4500 NC_001463GGGGGTAAAT CATTGGCAAG TGGATTATAC CCATTATGAA AATATCATAC  AF322109AGGAATAGAT CATTGGCAGG TAGACTATAC ACATTACAAA GAACACATAA4501                                              4550 NC_001463TATTAGTATG GGTAGAAACA AATTCAGGAC TAATATATGC AGAAAAAGTA  AF322109TATTAGTATG GGTAGAGACT AATTCAGGAT TAATATTTGC AGAGAAAGTA4551                                              4600 NC_001463AAAGGAGAAT CAGGGCAAGA ATTCAGAATA AAAGTGATGC ATTGGTATGC  AF322109AAAGGAGAAT CAGGACAAGA ATTTAGGATG CAGACATTGA AATGGTATGC4601                                              4650 NC_001463ATTATTTGGT CCAGAGTCAT TGCAGTCAGA CAATGGACCT GCATTTGCAG  AF322109TTTGTTTCAA CCAAAATCAG TGCAATCAGA TAATGGGACA GCCTTCACAG4651                                              4700 NC_001463CAGAGCCCAC ACAGCTGTTA ATGCAATACC TAGGAGTAAA ACACACAACA  AF322109CTGAGGCTAC GCAGCATCTA ATGAAGTATT TAGGGATTCA GCACACTACG4701                                              4750 NC_001463GGCATACCTT GGAATCCACA GTCTCAGGCT ATAGTAGAAA GGGCACATCA  AF322109GGTATTCCGT GGAACCCCCA GTCACAAAGT TTAGTAGAAA GAGCTCATCA4751                                              4800 NC_001463ACTATTGAAA AGCACTTTAA AGAAGTTCCA GCCACAATTT GTCGCTGTAG  AF322109AACATTAAAA CACATGTTAG AAAAATTAGA ACCACAATTT GTGGCCCTAC4801                                              4850 NC_001463AATCAGCCAT AGCAGCAGCC CTAGTCGCCA TAAATATAAA AAGAAAGGGT  AF322109AGTCTGCCAT CGCAGCCACT CTAGTTGCGC TCAATATAAA AAGAAAGGGT4851                                              4900 NC_001463GGGCTGGGGA CAAGCCCTAT GGATATTTTT ATATATAATA AAGAACAGAA  AF322109GGACTAGGGG CAAGCCCTAT GGATATTTAC ATATATAATA AGGAGCAACA4901                                              4950 NC_001463AAGAATAAAT AATAAATATA ATAAAAATTC TCAAAAAATT CAATTCTGTT  AF322109AAGACAACAA GATAATAGTA ATAAATTAAT TCAGAAAA.. .AATTTTGTT4951                                              5000 NC_001463ATTACAGAAT AAGGAAAAGA GGACATC.AG GAGAGTGGAA AGGACCAACC  AF322109ATTACAGGAT CAGAAAAAGA GGCCATCCAG GAGAGTGGAA CGGCCCAACT5001                                              5050 NC_001463CAGGTACTGT GGAAAGGGGA AGGAGCCAAT TGTGGTAAAG GATATAGAAA  AF322109GAGGTACTGT GGGAAGGGGA AGGAGCCA.T AGTAGTTAAA GACAAAGAAA5051                                              5100 NC_001463GTGAAAAGTA TTTAGTAATA CCTTACAAAG ATGCAAAATT CATCCCGCCA  AF322109GTGATAGATA TCTAGTCATC CCATATAAAG ATGCAAAATT TATTCCGCCA5101                                              5150 NC_001463CCAACAAAAG AAAAGGAATA AAAAACCTGG ACCAGAATTA CCCTTAGCAC  AF322109CCGTCGGAAC AGAAGGGATA GAAGAATAGG TCCAGAATTG CCTTTATCTT5151                                              5200 NC_001463TATGGATACA TATAGCAGAA AGCATTAATG GGGATAGCTC ATGGTACATA  AF322109TATGGACTTA TACAGCATAC AGCATAAATA AAGATCCCGC ATGGTATACA5201                                              5250 NC_001463ACAATGAGAC TGCAACAGAT GATGTGGGGA AAAAGAGGAA ATAAGTTACA  AF322109ACCCTAAGAC TGCAGCAAAT GATGTGGCAT AGGAGGGGAA ATAAATTGAC5251                                              5300 NC_001463ATATAAGAAT GAAGACAGGG AATATGAAAA TTGGGAAATT ACATCATGGG  AF322109ATATGTCAGG GAAAATGCAC AGTACGAGGA GTGGGAAATG ACCTCGTATG5301                                              5350 NC_001463GATGGAAAAT GCACCTAAGG AGAGTGAAAC AATGGATACA AGACAACAGG  AF322109AGTGGAGGAT AAGAATGAGA AGGGACAAAA CAAAAAGTCA TC.CAAGAGG5351                                              5400 NC_001463AGAGGAAGC. CCATGGCAGT ACAAAGTAGG AGGAACATGG AAAAGTATAG  AF322109GCATACTTCG CCATGGCAAT ATCGGAGACA GGATGGATGG AAGGATGTGG5401                                              5450 NC_001463GAGTGTGGTT CCTGCAAGCA GGAGATTACA GAAAGGTAGA CAGGCACTTC  AF322109GAACGTGGTT CCTACAGCCA GGGGACTATA GAAAGGCGGA TCAGCAGTTC5451                                              5500 NC_001463TGGTGGGCAT GGAGGATACT GATATGTTCC TGCAGGAAAG AAAAGTTTGA  AF322109TGGTTCGCTT GGAGAATAGT GTCGTGTTCA TGTAAAAAGG AAGGATTTAA5501                                              5550 NC_001463TATAAGAGAA TTTATGAGAG GAAGACATAG ATGGGATTTG TGCAAATCCT  AF322109CATAAGAGAA TTTATGCTAG GTACCCATAG ATGGGATTTG TGTAAGTCGT5551                                              5600 NC_001463GTGCTCAAGG AGAAGTAGTA AAGCATACTA GAACAAAAAG TCTGGAAAGA  AF322109GTTGCCAGGG TGAAGTAGTA AAGAGAACAC AACCCTACAC CTTGCAAAGG5601                                              5650 NC_001463CTAGTACTGC TACAGATGGT AGAACAGCAT GTGTTTCAAG TATTGCCATT  AF322109CTCACGTGGC TTAAATTAAC AGAAGACCAT GTATTTCAAG TAATGCCCTT5651                                              5700 NC_001463GTGGAGAGCC AGGAGAAGTA GTACAACAGA TTTCCCATGG TGCAGGGACA  AF322109GTGGAGAGCT CGCAAAGGGA TTACCATAGA CTTTCCCTGG TGCAGGGACA5701                                              5750 NC_001463CAACGGGATA CACGCATGCG TGGTCTGTCC AGGAGTGCTG GTTGATGGAA  AF322109CAAAAGGATT CCTGGAGCCG TGGACAACGC AAGAGTGTTG GCAAATAGAG5751                                              5800 NC_001463TATCTCTTAG AGGATGAGTG AAGAACTGCC TCAAAGAAGG GAGACACATC  AF322109TATCCCTTGG AGGATGAGTG AGGAAACCCC AGCAGGAAGA GAACCGACTG5801                                              5850 NC_001463CAGAAGAACT .TGTAAGGAA CGTACGGGAA AGAGAAAGGG ATACATGGCA  AF322109CAGAGGAAAT ATTTGAGCAA .......GAA GCAGAAAGT. .....TGGAA5851                                              5900 NC_001463ATGGACAAGC ATCAGAGTAC CTGCGGAAAT ACTGCAAAGA TGGCTTGCTA  AF322109GAGAACAAGC GTGCGAGTCC CAAATGACAT ATTACAAAGA TGGCTAGCAA5901                                              5950 NC_001463TGCTTAGGTC AGGCAGAAAT AGAAAGAAAG TGTATAGAGA AATGCAAAAA  AF322109TGCTTAGGCA AAGAGGAAAT AGAAAGAAAG TGCTTAGGGA AATGCAAAAA5951                                              6000 NC_001463TGGATGTGGA TACATCCCAA GGCGCCTGTG ATTAGGGCCT GTGGATGCAG  AF322109TGGGCATGGA GGAATCCCAC GGCGCGGGTG ATTCGGCCGT GTGGATGTCG6001                                              6050 NC_001463ACTATGTAAC CCGGGGTGGG GAACATAATC AAGGGAATAA TAAATGCAAA  AF322109GCTATGTAAC CCCGGCTGGG GGAG.TAATT AAT..CATAA TAAA.GCAAA6051                                              6100 NC_001463TAAATGTAAC TAACAAGTAG CAAAAGTGTC TGTGTTAGAT GGATGCTGGG  AF322109T...TGTAAC .......... .......... .......... ..ATGCTGTG6101                                              6150 NC_001463GCCAGATACA TGCGCTTAAC TGGGAAGGAA AACTGGGTTG AAGTAACCAT  AF322109TC.....A............. ..GG...... ...TGTCTTG CAGGAA...T6151                                              6200 NC_001463GGACGGAGAG AAGGAAAGGA AAAGAGAAGG TTTCACTGCG GGACAGCAAG  AF322109GG.CGGAGAT AAGAAAAG.. AA.GCAAAGG AGCCACT... ..AATCCAGG6201                                              6250 NC_001463GTAAGTATCA ACCCCAGGTA AGTAAGCAAA TAGGGAACAG AAATACTAAC  AF322109GTAAGTATAA AAAACAGGTA AGTA...... ...G...... AA....TAAC6251                                              6300 NC_001463CCATGCTTTG CCTATAAAGG GATATTCCTA TGGAGGATAT CACTAACAAT  AF322109.......... ....TATAGT TATATT.... ......A... ..CTAACAGT6301                                              6350 NC_001463GTGGATATTG CTAGGGATAA ATATGTGTGT CAGTGCAGAG GATTACATAA  AF322109AAGAGCAGCA CTAGG..... ..A....... ....GCAGAA ...TACATAA6351                                              6400 NC_001463CACTAATATC AGATCCCTAT GGGTTCTCAC CCATAAAAAA TGTGTCTGGG  AF322109CCATAATATC AGACCCATAT GGGTTCTCTC CCGTGAGAAA TGTGTCAGGA6401                                              6450 NC_001463GTACCAGTGA CTTGTGTAAC AAAAGAATTC GCAAAATGGG GATGTCAACC  AF322109GTACCTGTAA CTTGTGTGAC AAAAGAATTT AGTAAGTGGG GATGTCAGCC6451                                              6500 NC_001463ACTAGGAGCG TACCCTGATC CAGAAATAGA ATACAGAAAT GTGAGTCAGG  AF322109AATAGGAGCC TACCCAGACC CAGACTTAGA ATACAGAAAT ATAAGTAAAG6501                                              6550 NC_001463AAGTAGTGAA AGAAGTATAT CAAGAGAATT GGCCATGGAA TACATATCAT  AF322109AAATATTAGA GGAAGTATAT CAACAAGACT GGCCGTGGAA TACTTATCAT6551                                              6600 NC_001463TGGCCTCTCT GGCAAATGGA GAATGTTAGG TACTGGTTAA AAGAAAATAT  AF322109TGGCCATTAT GGCAAATGGA TAATGTAGTA CAATGGGCAA GGCAAAATTT6601                                              6650 NC_001463GCAAGAAAAT CAACAGAGAA AAAATAATAC AAAAGAGGGT ATAGAGGAAT  AF322109ACAGGATAAC CGCAAG.GAA AAAAG..... ......GGAC CTGGCAGACC6651                                              6700 NC_001463TATTAGCAGG AACTATAAGG GGAAGATTCT GTGTACCATA CCCATTTGCC  AF322109TATTAGCAGG AAAAATAAGG GGAAGATTCT GTGTACCCTA CCCATTTGCG6701                                              6750 NC_001463TTGTTAAAAT GCACAAAGTG GTGCTGGTAT ACAGCGGCCA TAAA..CAAC  AF322109CTCCTGGAGT GCATGGAATG GTGCTGGTGG GTTAAGAACA CTAATGCAGG6751                                              6800 NC_001463GAGTCA.GGA AAAGCAGGAA AAATAAAAAT AAATTGCACA GAAGCAAGAG  AF322109GGGGTATGGA GAAGCAG..A .CATAAGAAT AAATTGCTCA AGGGCAAGAG6801                                              6850 NC_001463CAGTCTCCTG TACAGAGGAC ATGCCATTAG CCTCAATACA AAGAGCATAT  AF322109CAGTGAGCTG CACAAGTGAA ATGCCCTTAG CATCCCTACA GAGGGTATAT6851                                              6900 NC_001463TGGGATGAGA AAGACAGAGA GAGCATGGCC TTTATGAATA TCAAAGCATG  AF322109TGGGAAAAGG AGGAACGAAA AAACATGGAG AAAATGACCA TCAAACCTTG6901                                              6950 NC_001463TGATAGCAAC CTAAGGTGTC AGAAAAGACC TGGAGGGTGT ATGGAAGGAT  AF322109CAATAAAAAT TTGGAATGCA AGAACAGAA. .G.GGGATGC GCAGAAGGGT6951                                              7000 NC_001463ACCCTATCCC AGTAGGAGCA GAAATAATCC CTGAAAGTAT GAAATACCTA  AF322109ATCCAGTACC TCCCAAGGCA GAGTTATTCC CTCCAGCGTT TCAGGATTTA7001                                              7050 NC_001463AGGGGAGCAA AGAGTCAG.. TATGGGGGAA TAAAAGATAA GAATGGAGAA  AF322109CAGCCA..AA AGGGTACGCA TATGGGGCAC TTAGAG...G GAACAGCAAA7051                                              7100 NC_001463TTAAAATTAC CATTAACATT AAGAGTGTGG GTAAAATTAG CAAATGTGTC  AF322109TTTCCACAAA GAGTGTCGCT AAGAACATGG GTGAAAATAG CTAACCTGAC7101                                              7150 NC_001463AGAATGGGTA AATGGGACAC CCCCGGATTG GCAAGACAGA ATTAACGGAT  AF322109AGGATGGGAA AAAGGAAAGC CAGCAGAATG GT......GG AATACCAG..7151                                              7200 NC_001463CCAAAGGAAT AAATGGGACG CTCTGGGGAG AGCTTAACAG TATGCATCAC  AF322109CCAACAGGTT CATTGGTTTG ATACCACGCC ACAATATCAT TTAGGAT...7201                                              7250 NC_001463CTAGGATTTG CCCTTAGCCA GAACGGCAAA TGGTGTAACT ACACCGGGGA  AF322109.ATGTATTAT CCCGAGCGCC TGAGAACAGG AGTTGTAATT TCACAGGGGA7251                                              7300 NC_001463AATAAAATTA GGGCAAGAAA CATTCCAATA TCATTACAAG CCAAACTGGA  AF322109AATACGAATA GGGCAACATC AGTTTGAGTA TAATTACACC CTGACAAAGA7301                                              7350 NC_001463ACTGTACC.. .GGGAATTGG ACGCAATATC CGGTGTGGCA AGTGATTAGA  AF322109ATTGCACAAA GGAGAAGTGG AAAGAGTACC CCATGTGGCA TGTCTGGAGG7351                                              7400 NC_001463AACCTGGATA TGGTGGAACA TATGACAGGA GAATGTGTGC AGAGACCACA  AF322109CATTTAGATC AAAATGAGCA CTTATCTAGC ATATGTTTCA AAAGACCGAG7401                                              7450 NC_001463AAGGCACAAT ATAACAGTAG GAAATGGAAC CATAACAGGG AATTGCAGTA  AF322109AAGAAATGCA ACACAAATAG GGAACAGTAC ACTGCAAGGG CAATGTAATA7451                                              7500 NC_001463CAACAAACTG GGATGGATGT AATTGCTCAC GATCAGGAAA CTACCTATAT  AF322109GAAGTAATTG GACAGGATGC CACTGCAATG AGACAGGGAT AAAC..AC..7501                                              7550 NC_001463AACAGCTCTG AGGGAGGATT GTTATTAATT CTGTGCAGAC AAAACAGCAC  AF322109AACA...... .......... .......... ...TGGAGAA TAAATGGCAC7551                                              7600 NC_001463CCTAACAAGG ATCCTGGGAA CAAATACAAA TTGGACAACT ATGTGGGGAA  AF322109....AAAGGG AGC.TT..AT CTCTTA..AA TAGCACTAAT .....GGAAA7601                                              7650 NC_001463TATACAAAAA TTGTTCAGGA TGCGAGAATG CAACATTAGA CAACACAGGA  AF322109CATCATGGTC TTGTT....A TGCTGGAACA CAACAGTGG. .....CAGGG7651                                              7700 NC_001463GAAGGAACCT TAGGAGGTGT AGCTAATAAG AACTGTAGCT TGCCTCATAA  AF322109GTA....... TATGAGAGTC AGCTAA.... A.GTGGAATG AGAGTCTTAA7701                                              7750 NC_001463AAATGAGAGC AACAAGTGGA CTTGTGCCCC AAGACAAAGA GATGGAAAAA  AF322109AGACGGAGAC TATGGGCTCT GTTTTAATTC AACAAACAGG AATTGTACTA7751                                              7800 NC_001463CAGATTC.GC TATACATAGC AGGAGGAAAA AAGTTTTGGA CACGAATTAA  AF322109GAAATGGAGC TCGGCACTAT GTAAACAAGA GAGTGATAAA AAACGAC.AC7801                                              7850 NC_001463GGCCCAATTC AGCTGTGAAA GTAACATAGG ACAATTAGAT GGAATGTTGC  AF322109AGCAGATCAT AATTGTGATA GCAGCATATC AGCAATAGAT GGAATGGTAC7851                                              7900 NC_001463ATCAGCAAAT ACTATTGCAA AAATATCAAG TAATTAAGGT AAGAGCTTAT  AF322109ATCAACAAAT ATTACTGCAA AGGTATCAAG TAATTAGAGT AAGAGCTTAC7901                                              7950 NC_001463ACATATGGGG TGATAGAAAT GCCAGAAAAC TATGCAAAAA CAAGAATCAT  AF322109ACATACGGAG TGATTGATAT GCCAGACAAT TATG.AGACC CTACCAGGA.7951                                              8000 NC_001463AAACAGGAAA AAAAGAGAAC TCAGCCACAA GAGGAAGAAG AGAGGCGTTG  AF322109....AGGAGA AGGAGAGATC TCGCAAAGGC CAGGAAAAAG AGGGGCGTGG8001                                              8050 NC_001463GCTTGGTCAT TATGCTAGTT ATCATGGCAA TAGTAGCTGC CGCAGGGGCT  AF322109GCCTGGTCAT CATGTTAGCT ATCATGGCCA TAGTGGCTGC TGCAGGAGCA8051                                              8100 NC_001463TCTCTGGGAG TCGCAAACGC GATTCAGCAG TCTTACACTA AGGCAGCTGT  AF322109TCTCTGGGAG TCGCGAACGC GATTCAGCAG TCCTACACCA GGGACGCTGT8101                                              8150 NC_001463CCAGACCCTT GCTAATGCAA CTGCTGCACA GCAGGATGTG TTAGAGGCAA  AF322109CCAGACTCTT GCTAACGCGA CTGCTGTGCA ACAGCAGGTG TTAGAGGCGT8151                                              8200 NC_001463CCTATGCCAT GGTACAGCAT GTGGCTAAAG GCGTACGAAT CTTGGAAGCT  AF322109CCTATGCCAT GATACAGCAT GTGGCTAAGG GAATACGCAT CCTTGAAGCA8201                                              8250 NC_001463CGAGTGGCTC GAGTGGAAGC TATCACAGAT AGAATAATGC TATACCAAGA  AF322109CGCGTGGCGA GAATGGAAGT TATGATGGAT AGAATGATGT TATATCAGGA8251                                              8300 NC_001463ATTGGATTGT TGGCACTATC ATCAATACTG TATAACCTCT ACAAAAACAG  AF322109AGTAGACTGC TGGCATTATC ACCAATATTG TGTAACCTCT ACAAGAGCAG8301                                              8350 NC_001463AAGTAGCAAA ATATATCAAT TGGACGAGGT TTAAGGATAA TTGCACATGG  AF322109ACATAGTGAA TTACATTAAT TGGACAAGGT TTAAAGATAA TTGCACATGG8351                                              8400 NC_001463CAGCAGTGGG AGAGAGGATT ACAGGGGTAT GATACAAACT TAACAATACT  AF322109CAAGAGTGGG AAAGGGAGAT AAGTGCGCAT GAAGGAAACA TCACTATATT8401                                              8450 NC_001463GTTAAAGGAA TCAGCAGCAA TGACACAACT AGCAGAAGAG CAAGCAAGGA  AF322109ACTCAAAGAA TCAGCAAGGA TAACACAATT AGCACAACAA AAGGTACAAA8451                                              8500 NC_001463GGATACCAGA AGTATGGGAA AGTTTAAAAG ACGTCTTTGA TTGGTCAGGA  AF322109GAATACCAGA TGTGTGGACA GCACTAAGGG AGTCACTAGG ATGGACACAA8501                                              8550 NC_001463TGGTTCTCAT GGCTAAAGTA TATTCCTATT ATAGTAGTAG GATTATTAGG  AF322109TGGCTGGCTT GGATAAAATA CCTTCCCATA ATAGTAGTAG GGATATTAGG8551                                              8600 NC_001463ATGCATTCTG ATAAGAGCTG TGATATGTGT ATGTCAACCT CTTGTGCAGA  AF322109ATGCATAATC ATAAGAATAA TGTTGTGTGT AGTACAACCA GTTCTTCAGA8601                                              8650 NC_001463TATACAGAAC TCTAAGTACC CCGACATACC AACGGGTCAC AGTCATCATG  AF322109TTTACAGAAC CTTGACTCAG ACCAGGTATC AACAAGTCAA CTTGGTGATG8651                                              8700 NC_001463GAAACAAGAG CAGACGTCGC AGGAGAAAAT CAGGATTTTG .....GATGG  AF322109GAGACCCGGG TGCAACTAGA AGAAGAAGAA GAAGAAGACG GAAGGGATGG8701                                              8750 NC_001463CTTAGAGGAA TCAGACAA.. .CAGCGAAAC AAGCGAAAGA GTGACAGTAC  AF322109TGGAGATGGC TCAGAGAGAT GCAGCGATCC CGACAACAAA .....AATTA8751                                              8800 NC_001463AGAAAGCTTG GAGCCGTGCC TGGGAGCTTT GGCAGAACTC ACCCTGGAAG  AF322109TGAACGCCTG GAGGAGAGCT TGGGTGACTT GGAGAAACTC ACCTTGGCAG8801                                              8850 NC_001463GAGCCATGGA AAAGGGGCCT GCTGAGGCTG CTCGTCCTTC CGCTGACGAT  AF322109AACACATGGA AGAATGTGGT GGTGGCGCCG TTGGTGATTC CGCTGACAAT8851                                              8900 NC_001463GGGAATCTGG ATAAATGGAT GGCTTGGAGA ACACCACAAA AATAAAAAAA  AF322109CAGAATTTGG CTCCTTGGAG AGAATGGAGA GAACCCCTAA AAGAAAAATA8901                                              8950 NC_001463GAAAGGGTG. ACTGTGAGAC ATGGGCTAAA GAGGACTAAT AACAAGCTAG  AF322109AAAAGGGTGG ACTGTGAGGA CTGTG..... .AGGCCTAGG AGCGAGATAG8951                                              9000 NC_001463GCCAAATTCC TGTAAATCAC TTGGGGGGTT ATAAGAAAAG CAAGTTCACT  AF322109...AAACTTA TAGGCCTCTC TTCCCGG... ......AAAG CTAACTCACT9001                                              9050 NC_001463ATGACAAAGC AAAATGTAAA GGCCAAATTC CTGTAAATCA CTTGGGGGGT  AF322109GTG....... .AGAGGAATA G..CAAGTCA CAGTGA..CA CT.....GCT9051                                              9100 NC_001463TATAAGAAAA GCAAGTTCAC TATGACAAAG CAAAATGTAA CCGCAAG...  AF322109AATTGTACCC GCAA...CCC TGAGATCATG CAAACCACAA TCCTGAGATT9101                                              9150 NC_004463.TGCTGACAG ATGTAACAGC TGACATATCA GCTGATGCTT GCTCATGCTG  AF322109ATGCTGACAT GTGTAACAGC TGATGCCTCA GCTGATGCTT GCTCATGCTG9151                                              9200 NC_001463ACACTGTAGC TCTGAGCTGT ATATAAGGAG AAGCTTGCTG CTTGC.ACTT  AF322109ACAATGTAAC TAGGAGCTCT ATATAAACAG AGCCCTAGAG CTTGCTACTT9201                                              9250 NC_001463CAGAGTTCTA GGAGAGTCCC .......... .TCCT.AGTC TCTCCTCTCC  AF322109CAGAGTGCTC TGAGGAGCTC GAAGGAAAGA GTCCTCAGCC TCTCCTCTCC9251                                              9300 NC_001463GAGGAGGTAC CGAGACCTCA AAATAAAGGA GTGATTGCCT TACTGCCGA.  AF322109GAGGAGCTTC GG....CTCA TAATAAAGGA GTGCTTGCTT CA..ACAGAA

TABLE 2 Pileup MSF: 759 Type: N Check: 1376 . . . Name: NC_001463(gag720 bp) (SEQ ID NO: 3) Len: 759 Check: 9060 Weight: 0 Name: AF322109 (gag720 bp) (SEQ ID NO: 4) Len: 759 Check: 2316 Weight: 0 //1                                                   50 NC_001463(gag720bp) ATGGTGAGTC TAGATAGAGA CATGGCGAGG CAAGTCTCCG GGGGGAAAAG AF322109 (gag720bp) .......... .......... .ATGGTGAGG CAGGCCTCCGGAAGGGGAAA 51                                                 100NC_001463 (gag720bp) AGATTATCCT GAGCTCGAAA AATGTATCAA GCATGCATGCAAGATAAAAG  AF322109 (gag720bp) GGAGTACCCC GAGCTAAAAG AATGTCTGAAAAAGGCATGC AAAATAAAAG101                                                150 NC_001463(gag720bp) TTCGACTCAG AGGGGAGCAC TTGACAGAAG GAAATTGTTT ATGGTGCCTT AF322109 (gag720bp) TAAGGGCTGG GGGGGAGCGC CTGACAGAAG GAAATTGTCTCTGGTGTATA 151                                                200NC_001463 (gag720bp) AAAACATTAG ATTACATGTT TGAGGACCAT AAAGAGGAACCTTGGACAAA  AF322109 (gag720bp) AAAACACTAG AGTGTATGTA TGAGGATTGTAGGGAGGAAC CTTGGACCCC201                                                250 NC_001463(gag720bp) AGTAAAATTT AGGACAATAT GGCAGAAGGT GAAGAATCTA ACTCCTGAGG AF322109 (gag720bp) AGAAAAATGT AAACAATTAT GGAAAAAGTT GAAGCAGGTAGAGCCTGAGG 251                                                300NC_001463 (gag720bp) AGAGTAACAA AAAAGACTTT ATGTCTTTGC AGGCCACATTAGCGGGTCTA  AF322109 (gag720bp) AGAGTAGCAA AGCAGACTAT AACTCGTTAAAAGCAACCTT GGCGGGGATA301                                                350 NC_001463(gag720bp) ATGTGTTGCC AAATGGGGAT GAGACCTGAG ACATTGCAAG ATGCAATGGC AF322109 (gag720bp) GTCTGTGTGC AAATGGGAAT GCAGCCCGAG ACACTGCAGGATGCGATAGC 351                                                400NC_001463 (gag720bp) TACAGTAATC ATGAAAGATG GGTTACTGGA ACAAGAGGAAAAGAAGGAAG  AF322109 (gag720bp) AACCTTAAAC ATGAGAGATG AAGT........AAAAGGAA AGGAA..AAG401                                                450 NC_001463(gag720bp) ACAAAAGAGA AAAGGAAGAG AGTGTCTTCC CAATAGTAGT GCAAGCAGCA AF322109 (gag720bp) CCATCAGAAG AAAAGAAGGG AATATAT..C CCATATTAGTGCAGGCAGGA 451                                                500NC_001463 (gag720bp) GGAGGGAGAA GCTGGAAAGC AGTAGATTCT GTAATGTTCCAGCAACTGCA  AF322109 (gag720bp) GGAGGAAGAG CATGGAGAGC GGTAGAGCCTGCTACCTTTC AGCAGCTCCA501                                                550 NC_001463(gag720bp) AACAGTAGCA ATGCAGCATG GCCTCGTGTC TGAGGACTTT GAAAGGCAGT AF322109 (gag720bp) AACAGTGGCA ATGCAGCATG GACTAGTATC AGAAGAATTTGAAAGGCAGC 551                                                600NC_001463 (gag720bp) TGGCATATTA TGCTACTACC TGGACAAGTA AAGACATACTAGAAGTATTG  AF322109 (gag720bp) TAGCATACTA TGCCACCACA TGGACAAGCAAGGATATCTT AGAAGTATTA601                                                650 NC_001463(gag720bp) GCCATGATGC CTGGAAATAG AGCTCAAAAG GAGTTAATTC AAGGGAAATT AF322109 (gag720bp) GCCATGATGC CAGGAAATAG AGCGCAAAAA GAACTAATACAAGGAAAGTT 651                                                700NC_001463 (gag720bp) AAATGAAGAA GCAGAAAGGT GGAGAAGGAA TAATCCACCACCTCCAGCAG  AF322109 (gag720bp) AAATGAGGAA GCAGAGAGAT GGAGAAGGCAGAATCCACAA CCTGCGG...701                                                750 NC_001463(gag720bp) GAGGAGGATT AACAGTGGAT .......... .......... .......... AF322109 (gag720bp) ...GCGGGTT AACCGTGGAT CAGATAATGG GGGTAGGACAAACGAATCAG 751 NC_001463 (gag720bp) .........  AF322109 (gag720bp)GCAGCGGCA Pileup MSF: 1347 Type: N Check: 2008 . . . Name: NC_001463(gag) (SEQ ID NO: 5) Len: 1347 Check: 6959 Weight: 0 Name:  AF322109(gag) (SEQ ID NO: 6) Len: 1347 Check: 5049 Weight: 0 //1                                                   50 NC_001463 (gag)ATGGTGAGTC TAGATAGAGA CATGGCGAGG CAAGTCTCCG GGGGGAAAAG  AF322109 (gag).......... .......... .ATGGTGAGG CAGGCCTCCG GAAGGGGAAA51                                                 100 NC_001463 (gag)AGATTATCCT GAGCTCGAAA AATGTATCAA GCATGCATGC AAGATAAAAG  AF322109 (gag)GGAGTACCCC GAGCTAAAAG AATGTCTGAA AAAGGCATGC AAAATAAAAG101                                                150 NC_001463 (gag)TTCGACTCAG AGGGGAGCAC TTGACAGAAG GAAATTGTTT ATGGTGCCTT  AF322109 (gag)TAAGGGCTGG GGGGGAGCGC CTGACAGAAG GAAATTGTCT CTGGTGTATA151                                                200 NC_001463 (gag)AAAACATTAG ATTACATGTT TGAGGACCAT AAAGAGGAAC CTTGGACAAA  AF322109 (gag)AAAACACTAG AGTGTATGTA TGAGGATTGT AGGGAGGAAC CTTGGACCCC201                                                250 NC_001463 (gag)AGTAAAATTT AGGACAATAT GGCAGAAGGT GAAGAATCTA ACTCCTGAGG  AF322109 (gag)AGAAAAATGT AAACAATTAT GGAAAAAGTT GAAGCAGGTA GAGCCTGAGG251                                                300 NC_001463 (gag)AGAGTAACAA AAAAGACTTT ATGTCTTTGC AGGCCACATT AGCGGGTCTA  AF322109 (gag)AGAGTAGCAA AGCAGACTAT AACTCGTTAA AAGCAACCTT GGCGGGGATA301                                                350 NC_001463 (gag)ATGTGTTGCC AAATGGGGAT GAGACCTGAG ACATTGCAAG ATGCAATGGC  AF322109 (gag)GTCTGTGTGC AAATGGGAAT GCAGCCCGAG ACACTGCAGG ATGCGATAGC351                                                400 NC_001463 (gag)TACAGTAATC ATGAAAGATG GGTTACTGGA ACAAGAGGAA AAGAAGGAAG  AF322109 (gag)AACCTTAAAC ATGAGAGATG ........AA GTAAAAGGAA AGGAA..AAG401                                                450 NC_001463 (gag)ACAAAAGAGA AAAGGAAGAG AGTGTCTTCC CAATAGTAGT GCAAGCAGCA  AF322109 (gag)CCATCAGAAG AAAAGAAGGG AATATATCCC ..ATATTAGT GCAGGCAGGA451                                                500 NC_001463 (gag)GGAGGGAGAA GCTGGAAAGC AGTAGATTCT GTAATGTTCC AGCAACTGCA  AF322109 (gag)GGAGGAAGAG CATGGAGAGC GGTAGAGCCT GCTACCTTTC AGCAGCTCCA501                                                550 NC_001463 (gag)AACAGTAGCA ATGCAGCATG GCCTCGTGTC TGAGGACTTT GAAAGGCAGT  AF322109 (gag)AACAGTGGCA ATGCAGCATG GACTAGTATC AGAAGAATTT GAAAGGCAGC551                                                600 NC_001463 (gag)TGGCATATTA TGCTACTACC TGGACAAGTA AAGACATACT AGAAGTATTG  AF322109 (gag)TAGCATACTA TGCCACCACA TGGACAAGCA AGGATATCTT AGAAGTATTA601                                                650 NC_001463 (gag)GCCATGATGC CTGGAAATAG AGCTCAAAAG GAGTTAATTC AAGGGAAATT  AF322109 (gag)GCCATGATGC CAGGAAATAG AGCGCAAAAA GAACTAATAC AAGGAAAGTT651                                                700 NC_001463 (gag)AAATGAAGAA GCAGAAAGGT GGAGAAGGAA TAATCCACCA CCTCCAGCAG  AF322109 (gag)AAATGAGGAA GCAGAGAGAT GGAGAAGGCA GAATCCACAA CCTGCGG...701                                                750 NC_001463 (gag)GAGGAGGATT AACAGTGGAT CAAATTATGG GGGTAGGACA AACAAATCAA  AF322109 (gag)...GCGGGTT AACCGTGGAT CAGATAATGG GGGTAGGACA AACGAATCAG751                                                800 NC_001463 (gag)GCAGCAGCAC AAGCTAACAT GGATCAGGCA AGGCAAATAT GCCTGCAATG  AF322109 (gag)GCAGCGGCAC AGGCTAATAT GGATCAAGCA AGACAAATAT GCCTACAATG801                                                850 NC_001463 (gag)GGTAATAAAT GCATTAAGAG CAGTAAGACA TATGGCGCAC AGGCCAGGGA  AF322109 (gag)GGTTATAACA GCAATAAGAG GAGTTAGGCA TATGGCCCAT AGACCAGGAA851                                                900 NC_001463 (gag)ATCCAATGCT AGTAAAGCAA AAAACGAATG AGCCATATGA AGATTTTGCA  AF322109 (gag)ATCCCATGCT GGTAAGACAA AAACCAAATG AGAACTATGA AGAGTTTGCC901                                                950 NC_001463 (gag)GCAAGACTGC TAGAAGCAAT AGATGCAGAG CCAGTTACAC AGCCTATAAA  AF322109 (gag)GCAAGGTTGT TAGAAGCAGT GGATGCAGAA CCCGTTACCC AACCTATAAA951                                               1000 NC_001463 (gag)AGATTATCTA AAGCTAACAC TATCTTATAC AAATGCATCA GCAGATTGTC  AF322109 (gag)AGAATATTTA AAGGTAACTC TGTCTTACAC AAATGCAAAT TCGGAATGTC1001                                              1050 NC_001463 (gag)AGAAGCAAAT GGATAGAACA CTAGGACAAA GAGTACAACA AGCTAGTGTA  AF322109 (gag)AAAAACATAT GGACAGAGTG TTGGGGCAAA GAGTACAGCA GGCCTCAATA1051                                              1100 NC_001463 (gag)GAAGAAAAAA TGCAAGCATG TAGAGATGTG GGATCAGAAG GGTTCAAAAT  AF322109 (gag)GAAGAAAAAA TGCAGGCATG CAGGGACATC GGGGGAACAG CATATCAGAT1101                                              1150 NC_001463 (gag)GCAATTGTTA GCACAAGCAT TAAGGCCAGG AAAAGGAAAA GGGAATGGAC  AF322109 (gag)GCAGTTGCTT GCACAAGCCC TCCGTGGCGG AAAAGAAGAT GGGAAAAAAT1151                                              1200 NC_001463 (gag)AGCCACAAAG GTGTTACAAC TGTGGAAAAC CGGGACATCA AGCAAGGCAA  AF322109 (gag)CTGTAGGGAA GTGTTATAAC TGTGGAAGGC CCGGACACAG AGCAAAAGAA1201                                              1250 NC_001463 (gag)TGTAGACAAG GAATCATATG TCACAACTGT GGAAAGAGAG GACATATGCA  AF322109 (gag)TGCAGACAAG GCATTATATG TCACAACTGT GGAAAAAGAG GGCATATACA1251                                              1300 NC_001463 (gag)AAAAGAATGC AGAGGAAAGA GAGACATAAG GGGAAAACAG CAGGGAAACG  AF322109 (gag)GAAAAACTGC A.....AACA GAAA....AG AAGAAAGGAG CAGGGAAACA1301                                           1347 NC_001463 (gag)GGAGGAGGGG GATACGTGTG GTGCCGTCCG CTCCTCCTAT GGAATAA  AF322109 (gag)TGAGGAGGGG GCTACGTGTG GTGCCGTCCG CACCCCCTAT GGAGTAA

TABLE 3 PileUp MSF: 605 Type: N Check: 9138 . . . Name: NC_001463 (5′)(SEQ ID NO: 7) Len: 605 Check: 5398 Weight: 0 Name:  AF322109 (5′) (SEQID NO: 8) Len: 605 Check: 3740 Weight: 0 //1                                                   50 NC_001463 (5′).......... ....GAGTTC TAGG...ACA GTCCCTCCTA GTCTCTCCTC  AF322109 (5′)GTGAGTGCTC TGAGGAGCTC GAAGGAAAGA GTCC.TC..A GCCTCTCCTC51                                                 100 NC_001463 (5′)TCCGAGGAGG TACCGAGACC TCAAAATAAA GGAGTGATTG CCTTACTGCC  AF322109 (5′)TCCGAGGAGC TTCGG....C TCATAATAAA GGAGTGCTTG CTTCA..ACA101                                                150 NC_001463 (5′)GAGTGGAGAG TGATTACTGA GCGGCCGGTG TATCGGGAGT CGTCCCTTAA  AF322109 (5′)GAACTGAG.. ......CTGG TCGTGGTTAT TATCGGG... .GACCGAAGT151                                                200 NC_001463 (5′)TCTGTGCAAT ACCAGAGCGG CTCTCGCAGC TGGCGCCCAA CGTGGGGCCC  AF322109 (5′)CCCGTGCAAC ACCGGGGCGG TTCTCGCAGC TGGCGCCCAA CGTGGGGCTC201                                                250 NC_001463 (5′)GAGGAG.... .......... .......... .......... ..........  AF322109 (5′)GAGTAGCTTG AGAAGCTCGA CTGAGATCTG AATCCAAGAG CGACATCAGA251                                                300 NC_001463 (5′)....AAGAAA AGAAAGC... GGCCCTGAGA ACTCGGCTTC TG..AAAAAG  AF322109 (5′)CAGCAAGAAA TGAGAGTAAT GAGACCGCGA GCTCTGCTGC TGTAAAAAAG301                                                350 NC_001463 (5′)AGGAAGAGGA CAAGTTGCTA TAGCAACAAG AGAGAAGAAG TAGAGCAAAG  AF322109 (5′)AGGAAGTAG. CGGGTTGCCG AGGCAACTGC TCAGAAGAAC CAGGGGAAAG351                                                400 NC_001463 (5′)GTCCAGTGGC T.CGGAAAAA GAGGAACTGA AACTTCGGGG ACGCCTGAAG  AF322109 (5′)GGCTTCCAGC AACCTCAAAA GAGGAACCGA GACTTCGGGG ACGCCTGAA.401                                                450 NC_001463 (5′)GAGTAAGGTA AGTGACTCTG CTGTACGCGG GGCGAGGCAG AGGTT.TCCT  AF322109 (5′)..GTAAGGTA AGTGACTCTG CTGTACGCGG GGCGAGGCAT AGGAGATCCT451                                                500 NC_001463 (5′)TCTAAATT.G AAAGAGAAGT GTTGCTGCGA GAGGTCTTGG TGGTCGAGAA  AF322109 (5′)TCTATTCTAG GAAGAGAAGC GCTGTTCTGG GAGGTCTTGG CGACCGAGAA501                                                550 NC_001463 (5′)TCCTGTACAA AAAAAAGGAG GGATCTCGGT CAGGACCAGG ACCCCTGGGA  AF322109 (5′)TCTTGTT... AAATAAGCCA GGATCTCGAT CAGGACCAAG ACCCCTCAGG551                                                600 NC_001463 (5′)GTAATACAAC AGCAACACCG TAAGAAAATC CGCC...... ..........  AF322109 (5′)AGAGGGTATA GACAGCGTGG TAAGAAA.TC CGCCGTGGTG AGTCTAGATA 601 NC_001463(5′) .....  AF322109 (5′) GAGAC

TABLE 4 Pileup MSF: 3338 Type: N Check: 5428 . . . Name: NC_001463 (pol)(SEQ ID NO: 9) Len: 3338 Check: 8114 Weight: 0 Name:  AF322109 (pol)(SEQ ID NO: 10) Len: 3338 Check: 7314 Weight: 0 //1                                                   50 NC_001463 (pol).......... ........AT GTCACAACTG TGGAAAGAGA GGACATATGC  AF322109 (pol)ATGCAGACAA GGCATTATAT GTCACAACTG TGGAAAAAGA GGGCATATAC51                                                 100 NC_001463 (pol)AAAAAGAATG CAGAGGAAAG AGAGACATAA GGGGAAAACA GCAGGGAAAC  AF322109 (pol)AGAAAAACTG CA.....AAC AGAAA....A GAAGAAAGGA GCAGGGAAAC101                                                150 NC_001463 (pol)GGGAGGAGGG GGATACGTGT GGTGCCGTCC GCTCCTCCTA TGGAATAACT  AF322109 (pol)ATGAGGAGGG GGCTACGTGT GGTGCCGTCC GCACCCCCTA TGGAGTAACG151                                                200 NC_001463 (pol)TCAGCACCAC CTATGGTTCA GGTCCGCATA GGTTCCCAGC AGAGGAACTT  AF322109 (pol)CAAGCACCAC TAATAGTTAG GGTACAAATA CGGAATCAGG AGAAACAATT201                                                250 NC_001463 (pol)GTTATTTGAT ACCGGGGCGG ACCGAACTAT AGTTAGATGG CATGAGGGCT  AF322109 (pol)ATTATTTGAC ACAGGGGCAG ATAAAACGAT AGTAAGAATG CATGATGGAA251                                                300 NC_001463 (pol)CGGGAAACCC AGCCGGAAGG ATAAAACTGC AAGGAATAGG AGGAATAGTA  AF322109 (pol)CAGGGATTCC AAACGGAAGA ATAAAATTAC AAGGGATAGG AGGAATAGTA301                                                350 NC_001463 (pol)GAAGGAGAAA AATGGAATAA TGTAGAATTA GAATATAAAG GAGAAACAAG  AF322109 (pol)GAAGGAGAAA AATGGAATAA AGTACCCATG ACATATAAGG GAGAAACATC351                                                400 NC_001463 (pol)AAAGGGAACA ATAGTAGTGT TACCACAAAG TCCAGTAGAA GTATTAGGAC  AF322109 (pol)CTGCCCAAGC TTGGTTGTGC TAAGAGATAG CCCAGTAGAA GTATTGGGAA401                                                450 NC_001463 (pol)GAGATAACAT GGCCCGATTT GGAATAAAGA TAATAATGGC AAATTTAGAG  AF322109 (pol)GAGATAACAT GGAAGCATTC GGCGTAACCC TAATAATGOC AAATTTAGAA451                                                500 NC_001463 (pol)GAAAAAAGAA TCCCAATTAC AAAAGTAAAA TTGAAAGAGG GATGTACGGG  AF322109 (pol)GATAAGAAAA TTCCCACAAT ACCAGTAGAA TTGAAAGAAG GATGTAAAGG501                                                550 NC_001463 (pol)TCCACATGTC CCACAATGGC CATTAACAGA AGAGAAATTA AAAGGTCTAA  AF322109 (pol)GCCACATGTG CCCCAGTGGC CATTAACAGC AGAGAAATTA CAAGGACTAA551                                                600 NC_001463 (pol)CAGAAATCAT AGATAAATTA GTGGAAGAAG GAAAACTAGG AAAGGCACCC  AF322109 (pol)CAGGAATAGT AGAAAAATTA CTACAGGAAG GAAAATTGGC AGAGGCCCCA601                                                650 NC_001463 (pol)CCACATTGGA CATGTAATAC TCCAATCTTT TGCATAAAAA AGAAATCAGG  AF322109 (pol)GAGGGATGGA CGTGGAACAC GCCCATCTTC TGCATAAAAA AGAAGTCAGG651                                                700 NC_001463 (pol)GAAGTGGAGA ATGTTAATAG ATTTCAGAGA ATTGAACAAA CAGACAGAAG  AF322109 (pol)AAAATGGAGA ATGTTAATAG ATTTTAGGGA ATTAAATAAG CAAACAGCAG701                                                750 NC_001463 (pol)ATTTAACAGA AGCGCAGTTA GGACTCCCGC ATCCGGGAGG ACTACAAAAG  AF322109 (pol)ATTTAGCAGA AGCGCAGCTA GGACTGCCAC ACCCAGGAGG GTTGCAAAGG751                                                800 NC_001463 (pol)AAAAAACATG TTACAATATT GGACATAGGA GATGCATATT TTACTATACC  AF322109 (pol)AAAAAGAATG TAACAATTCT GGACATAGGA GATGCATATT TCACAATTCC801                                                850 NC_001463 (pol)CCTATATGAA CCATATCGAG AGTACACATG TTTTACTCTA TTAAGTCCTA  AF322109 (pol)CTTATACGAG CCCTATCAGA AATATACATG CTTCACACTC CTAAGTCCTA851                                                900 NC_001463 (pol)ATAATCTAGG ACCATGTAAA AGATACTATT GGAAAGTGCT GCCACAAGGT  AF322109 (pol)ACAATTTGGG ACCATGTAAA AGGTATTATT GGAAAGTATT ACCCCAGGGA901                                                950 NC_001463 (pol)TGGAAATTGA GTCCATCTGT ATATCAATTT ACTATGCAGG AGATCTTAGA  AF322109 (pol)TGGAAATTGA GCCCAGCTGT ATATCAATTC ACCATGCAAA GGTTGTTAAA951                                               1000 NC_001463 (pol)GGATTGGATA CAGCAGCATC CAGAAATTCA ATTTGGCATA TATATGGATG  AF322109 (pol)AGGATGGATA CAACAGCATA AAAACATACA ATTTGGAATA TATATGGATG1001                                              1050 NC_001463 (pol)ATATTTACAT AGGAAGTGAT TTAGAAATTA AAAAGCATAG AGAAATAGTG  AF322109 (pol)ATATCTATAT TGGAAGTGAT CTAACGATAG CCCAACATAG GAAGATAATA1051                                              1100 NC_001463 (pol)AAAGATTTAG CCAATTATAT TGCCCAATAT GGATTCACTC TGCCAGAAGA  AF322109 (pol)GAAGAATTAG CCTCATTTAT AGAACAATTT GGGTTTACAT TACCAGAAGA1101                                              1150 NC_001463 (pol)GAAGAGACAA AAGGGATATC CAGCAAAATG GCTAGGATTT GAACTACACC  AF322109 (pol)TAACAGACAA GAGGGCTATC CAGCAAAATG GCTAGGATTC GAGCTACATC1151                                              1200 NC_001463 (pol)CGCAGACCTG GAAATTTCAG AAGCATACAT TACCTGAATT AACAAAGGGA  AF322109 (pol)CAGAAAAATG GAAATATCAA AAGCATAAAT TGCCGGAATT ACAAGAGGGG1201                                              1250 NC_001463 (pol)ACAATAACAT TAAATAAATT ACAGAAATTA GTAGGAGAAT TAGTATGGAG  AF322109 (pol)GTAATAACCC TGAACAAATT ACAGAAGATA GTAGGGGAAT TAGTGTGGAG1251                                              1300 NC_001463 (pol)ACAATCCATA ATTGGGAAAA GCATTCCTAA CATTCTGAAA TTAATGGAAG  AF322109 (pol)ACAATCCTTG ATAGGAAAGA GCATCCCCAA TATCATAAAA TTAATGGAAG1301                                              1350 NC_001463 (pol)GAGATAGAGA ATTACAAAGT GAAAGAAAAA TTGAAGAAGT ACATGTGAAA  AF322109 (pol)GAGATCGCGC ATTACAAAGT GAAAGGAAAA TAGAAAGAAT ACATGTACAA1351                                              1400 NC_001463 (pol)GAATGGGAAG CATGTAGGAA AAAATTAGAA GAAATGGAAG GAAATTATTA  AF322109 (pol)GAATGGGAAG CATGTCAAAA GAAATTAGAT GAAATGGTAG GAAATTATTA1401                                              1450 NC_001463 (pol)TAATAAAGAC AAAGATGTCT ATGGACAATT GGCTTGGGGA GACAAAGCTA  AF322109 (pol)CAGAGAAGAA GAAGATATCT ATGGACAAAT AACTTGGGGG GATAAGGCAA1451                                              1500 NC_001463 (pol)TAGAATATAT AGTGTATCAG GAGAAAGGGA AACCATTATG GGTAAATGTG  AF322109 (pol)TAAAATACAT AGTATTCCAA AGGAAAGGGG AACCCCTATG GGTAAATGTA1501                                              1550 NC_001463 (pol)GTTCACAATA TAAAGAACCT AAGCATCCCG CAACAGGTTA TTAAAGCAGC  AF322109 (pol)GTACATGACA TAAAAAATTT GAGTCTCCCA CAGCAAGTGA TAAAAGCAGC1551                                              1600 NC_001463 (pol)GCAAAAATTA ACCCAAGAAG TCATCATTAG GACAGGAAAA ATACCATGGA  AF322109 (pol)ACAGAAATTA ACCCAGGAAG TAATCATAAG AACAGGAAAA ATCCCATGGC1601                                              1650 NC_001463 (pol)TATTGTTGCC AGGGAAAGAA GAAGATTGGA GACTAGAATT GCAATTAGGG  AF322109 (pol)TGCTACTACC AGGAAGAGAA GAAGACTGGA GATTAGAACT GCAGGTAGGG1651                                              1700 NC_001463 (pol)AACATCACAT GGATGCCAAA ATTTTGGTCC TGTTATCGAG GA.CATACAA  AF322109 (pol)AACATCACGT GGATGCCATC ATTTTGGTCA TGTTATCGAG GAGCACCCAA1701                                              1750 NC_001463 (pol)GATGGAGAAA AAGAAATATA ATAGAAGAAG TAGTAGAAGG GCCTACATAT  AF322109 (pol)G.TGGAAAAG AAGGAACATA GTGGCAGCAG TGGTAGATGG ACCCACATAT1751                                              1800 NC_001463 (pol)TATACAGATG GAGGAAAAAA GAATAAAGTA GGAAGTCTAG GGTTCATAGT  AF322109 (pol)TATACAGATG GGGGAAAGAA AAACGCACAG GGAAGCTTTG GCTTCATCTC1801                                              1850 NC_001463 (pol)ATCAACAGGG GAAAAATTTA GAAAGCATGA AGAGGGCACA AACCAGCAAC  AF322109 (pol)CCCAACAGGA GAAAAGTTCA GAAGGCATGA AGATGGAACT AATCAGGTAT1851                                              1900 NC_001463 (pol)TAGAATTAAG AGCCATAGAG GAAGCTCTAA AACAAGGGCC TCAAACAATG  AF322109 (pol)TAGAATTAAG GGCAATAGAA GATCCATGTA AACAAGGACC TGAAAGCATG1901                                              1950 NC_001463 (pol)AATTTAGTAA CAGATAGTAG ATATGCATTT GAATTTTTAT TAAGAAATTG  AF322109 (pol)AACATTGTAA CTGACAGCAG GTATGCTTAT GAATTCATGC TCCGAAACTG1951                                              2000 NC_001463 (pol)GGATGAAGAA GTAATAAAGA ATCCAATTCA AGCAAGAATT ATGGAAATTG  AF322109 (pol)GGATGAACAG GTCATAAGAA ACCCCATTCA GGCAAGAATC ATGGCAGAAG2001                                              2050 NC_001463 (pol)CCCACAAGAA AGATAGGATA GGAGTGCATT GGGTGCCAGG ACATAAAGGG  AF322109 (pol)TGCACAAGAA AAAGCAGGTA GGAATACACT GGGTGCCAGG GCATAAAGGA2051                                              2100 NC_001463 (pol)ATTCCCCAAA ATGAAGAAAT AGACAAATAT ATTTCGGAAA TATTTCTTGC  AF322109 (pol)ATACCTCAGA ATGAAGAGAT AGACCAGTAC ATATCAGAAG TATTCTTAGC2101                                              2150 NC_001463 (pol)AAAAGAAGGA GAAGGAATTC TCCCAAAAAG AGAAGAGGAT GCAGGGTATG  AF322109 (pol)ACGAGAAGGA ACAGGGATAT GTGAAAAAAG GAAGGAAGAT GCTGGATATG2151                                              2200 NC_001463 (pol)ATTTAATATG CCCAGAAGAG GTTACCATAG AGCCAGGACA AGTGAAATGC  AF322109 (pol)ATTTATTATG CCCGCATGAG GTAATACTTA AACCCCAAGA AGTAAAACGG2201                                              2250 NC_001463 (pol)ATCCCCATAG AGCTAAGATT AAATTTAAAG AAATCACAAT GGGCTATGAT  AF322109 (pol)ATCCCAATAG ACCTAAAATT AAAATTGAAA GAAAAGCAAT GGGCCATGAT2251                                              2300 NC_001463 (pol)TGCTACAAAA AGCAGCATGG CTGCCAAAGG AGTGTTCACA CAAGGAGGAA  AF322109 (pol)AAGTGGGAAA AGTAGCGTTG CAGCAAAAGG AATATTTGTA CAAGGAGGCA2301                                              2350 NC_001463 (pol)TCATAGACTC AGGATATCAG GGACAAATAC AGGTAATAAT GTATAATAGC  AF322109 (pol)TAATAGATTC AGGGTATCAG GGACAAGTAC AAGTCATCCT ATATAATAGT2351                                              2400 NC_001463 (pol)AATAAAATAG CAGTAGTCAT ACCCCAAGGG AGAAAATTTG CACAATTAAT  AF322109 (pol)AATAAGATAG AGGTCAAAAT ACCACAAGGC AGGAAATTTG CCCAATTAAT2401                                              2450 NC_001463 (pol)ATTAATGGAT AAAAAGCATG GAAAATTGGA ACCCTGGGGG GAAAGCAGAA  AF322109 (pol)ATTAATGAAC TTACAACATG AAGAATTAGA AGAATGGGGA AAGGAAAGAA2451                                              2500 NC_001463 (pol)AAACAGAAAG GGGAGAAAAA GGATTTGGGT CTACAGGAAT GTATTGGATA  AF322109 (pol)AAACAGAAAG AGGAACAAAA GGATTTGGGT CTACAGGAGC ATTTTGGATA2501                                              2550 NC_001463 (pol)GAAAATATTC CTCTGGCAGA GGAAGACCAC ACAAAATGGC ATCAAGATGC  AF322109 (pol)GAGAATATTC CCCAAGCAGA GGAAGAACAT TACAAATGGC ATCAAGATGC2551                                              2600 NC_001463 (pol)CCGATCATTG CATCTAGAAT TTGAAATTCC AAGAACAGCA GCAGAAGACA  AF322109 (pol)TAGATCTCTG CAGCTAGAAT TCAAGATACC TAGAGCAGCA GCAGAAGACA2601                                              2650 NC_001463 (pol)TAGTAAATCA ATGTGAAATA TGCAAAGAAG CGAGGACACC TGCAGTAATT  AF322109 (pol)TTATACAGCA CTGTGAGGTA TGTCAAGAAG GCAAACCCGC AGCGATCACG2651                                              2700 NC_001463 (pol)AGAGGCGGAA ACAAAAGGGG GGTAAATCAT TGGCAAGTGG ATTATACCCA  AF322109 (pol)AGAGGGGGAA ATAAAAGAGG AATAGATCAT TGGCAGGTAG ACTATACACA2701                                              2750 NC_001463 (pol)TTATGAAAAT ATCATACTAT TAGTATGGGT AGAAACAAAT TCAGGACTAA  AF322109 (pol)TTACAAAGAA CACATAATAT TAGTATGGGT AGAGACTAAT TCAGGATTAA2751                                              2800 NC_001463 (pol)TATATGCAGA AAAAGTAAAA GGAGAATCAG GGCAAGAATT CAGAATAAAA  AF322109 (pol)TATTTGCAGA GAAAGTAAAA GGAGAATCAG GACAAGAATT TAGGATGCAG2801                                              2850 NC_001463 (pol)GTGATGCATT GGTATGCATT ATTTGGTCCA GAGTCATTGC AGTCAGACAA  AF322109 (pol)ACATTGAAAT GGTATGCTTT GTTTCAACCA AAATCAGTGC AATCAGATAA2851                                              2900 NC_001463 (pol)TGGACCTGCA TTTGCAGCAG AGCCCACACA GCTGTTAATG CAATACCTAG  AF322109 (pol)TGGGACAGCC TTCACAGCTG AGGCTACGCA GCATCTAATG AAGTATTTAG2901                                              2950 NC_001463 (pol)GAGTAAAACA CACAACAGGC ATACCTTGGA ATCCACAGTC TCAGGCTATA  AF322109 (pol)GGATTCAGCA CACTACGGGT ATTCCGTGGA ACCCCCAGTC ACAAAGTTTA2951                                              3000 NC_001463 (pol)GTAGAAAGGG CACATCAACT ATTGAAAAGC ACTTTAAAGA AGTTCCAGCC  AF322109 (pol)GTAGAAAGAG CTCATCAAAC ATTAAAACAC ATGTTAGAAA AATTAGAACC3001                                              3050 NC_001463 (pol)ACAATTTGTC GCTGTAGAAT CAGCCATAGC AGCAGCCCTA GTCGCCATAA  AF322109 (pol)ACAATTTGTG GCCCTACAGT CTGCCATCGC AGCCACTCTA GTTGCGCTCA3051                                              3100 NC_001463 (pol)ATATAAAAAG AAAGGGTGGG CTGGGGACAA GCCCTATGGA TATTTTTATA  AF322109 (pol)ATATAAAAAG AAAGGGTGGA CTAGGGGCAA GCCCTATGGA TATTTACATA3101                                              3150 NC_001463 (pol)TATAATAAAG AACAGAAAAG AATAAATAAT AAATATAATA AAAATTCTCA  AF322109 (pol)TATAATAAGG AGCAACAAAG ACAACAACAT AATAGTAATA AATTAATTCA3151                                              3200 NC_001463 (pol)AAAAATTCAA TTCTGTTATT ACAGAATAAG GAAAAGAGGA CATC.AGGAG  AF322109 (pol)GAAAA...AA TTTTGTTATT ACAGGATCAG AAAAAGAGGC CATCCAGGAG3201                                              3250 NC_001463 (pol)AGTGGAAAGG ACCAACCCAG GTACTGTGGA AAGGGGAAGG AGCCAATTGT  AF322109 (pol)AGTGGAACGG CCCAACTGAG GTACTGTGGG AAGGGGAAGG AGCCA.TAGT3251                                              3300 NC_001463 (pol)GGTAAAGGAT ATAGAAAGTG AAAAGTATTT AGTAATACCT TACAAAGATG  AF322109 (pol)AGTTAAAGAC AAAGAAAGTG ATAGATATCT AGTCATCCCA TATAAAGATG3301                                 3338 NC_001463 (pol) CAAAATTCATCCCGCCACCA ACAAAAGAAA AGGAATAA  AF322109 (pol) CAAAATTTAT TCCGCCACCGTCGGAACAGA AGGGATAG

TABLE 5 Pileup MSF: 408 Type: N Check: 517 . . . Name: NC_001463 (rev)(SEQ ID NO: 11) Len: 408 Check: 7287 Weight: 0 Name:  AF322109 (rev)(SEQ ID NO: 12) Len: 408 Check: 3230 Weight: 0 //1                                                   50 NC_001463 (rev)ATGGATGCTG GGGCCAGATA CATGCGCTTA ACTGGGAAGG AAAACTGGGT  AF322109 (rev).......... .......... .......... .......... ..........51                                                 100 NC_001463 (rev)TGAAGTAACC ATGGACGGAG AGAAGGAAAG GAAAAGAGAA GGTTTCACTG  AF322109 (rev).......... ATGG.CGGAG ATAAGAAAAG ..A.AGCAAA GGAGCCACTA101                                                150 NC_001463 (rev)CGGGACAGCA AGATATACAG AACTCTAAGT ACCCCGACAT ACCAACGGGT  AF322109 (rev)ATCCAGGACC AGGTATCAAC AAGTCAACTT GGTGATGGAG ACC..CGGGT151                                                200 NC_001463(rev)CACAGTCATC ATGGAAACAA GAGCAGACGT CGCAGGAGAA AATCAGGATT  AF322109 (rev).......... ..GCAACTAG AAGAAGAAGA AGAAGAAGAC GGAAGGGATG201                                                250 NC_001463 (rev)TTGGCGATGG CTTAGAGGAA TCAGACAACA GCGAAACAAG CGAAAGAGTG  AF322109 (rev)GTGGAGATGG CTCAGAGAGA TG......CA GCGATCCCGA CAACAAAGGA251                                                300 NC_001463 (rev)ACAGTACAGA AAGCTTGGAG CCGTGCCTGG GAGCTTTGGC AGAACTCACC  AF322109 (rev)A...TTATGA ACGCCTGGAG GAGAGCTTGG GTGACTTGGA GAAACTCACC301                                                350 NC_001463 (rev)CTGGAAGGAG CCATGGAAAA GGGGCCTGCT GAGGCTGCTC GTCCTTCCGC  AF322109 (rev)TTGGCAGAAC ACATGGAAGA ATGTGGTGGT GGCGCCGTTG GTGATTCCGC351                                                400 NC_001463 (rev)TGACGATGGG AATCTGGATA AATGGATGGC TTGGAGAACA CCACAAAAAT  AF322109 (rev)TGACAATCAG AATTTGGCTC CTTGGAGAGA ATGGAGAGAA CCCCTAAAAG 401 NC_001463(rev) AA......  AF322109 (rev) AAAAATAA

TABLE 6 Pileup MSF: 691 Type: N Check: 6528 . . . Name: NC_001463 (vif)(SEQ ID NO: 13) Len: 691 Check: 5882 Weight: 0 Name:  AF322109 (vif)(SEQ ID NO: 14) Len: 691 Check:  646 Weight: 0 //1                                                   50 NC_001463 (vif)ATGCAAAATT CATCCCGCCA CCAACAAAAG AAAAGGAATA AAAAACCTGG  AF322109 (vif)ATGCAAAATT TATTCCGCCA CCGTCGGAAC AGAAGGGATA GAAGAATAGG51                                                 100 NC_001463 (vif)ACCAGAATTA CCCTTAGCAC TATGGATACA TATAGCAGAA AGCATTAATG  AF322109 (vif)TCCAGAATTG CCTTTATCTT TATGGACTTA TACAGCATAC AGCATAAATA101                                                150 NC_001463 (vif)GGGATAGCTC ATGGTACATA ACAATGAGAC TGCAACAGAT GATGTGGGGA  AF322109 (vif)AAGATCCCGC ATGGTATACA ACCCTAAGAC TGCAGCAAAT GATGTGGCAT151                                                200 NC_001463 (vif)AAAAGAGGAA ATAAGTTACA ATATAAGAAT GAAGACAGGG AATATGAAAA  AF322109 (vif)AGGAGGGGAA ATAAATTGAC ATATGTCAGG GAAAATGCAC AGTACGAGGA201                                                250 NC_001463 (vif)TTGGGAAATT ACATCATGGG GATGGAAAAT GCACCTAAGG AGAGTCAAAC  AF322109 (vif)GTGGGAAATG ACCTCGTATG AGTGGAGGAT AAGAATGAGA AGGGACAAAA251                                                300 NC_001463 (vif)AATGGATACA AGACAACAGG AGAGGAAGC. CCATGGCAGT ACAAAGTAGG  AF322109 (vif)CAAAAAGTCA TC.CAAGAGG GCATACTTCG CCATGGCAAT ATCGGAGACA301                                                350 NC_001463 (vif)AGGAACATGG AAAAGTATAG GAGTGTGGTT CCTGCAAGCA GGAGATTACA  AF322109 (vif)GGATGGATGG AAGGATGTGG GAACGTGGTT CCTACAGCCA GGCGACTATA351                                                400 NC_001463 (vif)GAAAGGTAGA CAGGCACTTC TGGTGGGCAT GGAGGATACT GATATGTTCC  AF322109 (vif)GAAAGGCGGA TCAGCAGTTC TGGTTCGCTT GGAGAATAGT GTCGTGTTCA401                                                450 NC_001463 (vif)TGCAGGAAAG AAAAGTTTGA TATAAGAGAA TTTATGAGAG GAAGACATAG  AF322109 (vif)TGTAAAAAGG AAGGATTTAA CATAAGAGAA TTTATGCTAG GTACCCATAG451                                                500 NC_001463 (vif)ATGGGATTTG TGCAAATCCT GTGCTCAAGG AGAAGTAGTA AAGCATACTA  AF322109 (vif)ATGGGATTTG TGTAAGTCGT GTTGCCAGGG TGAAGTAGTA AAGAGAACAC501                                                550 NC_001463 (vif)GAACAAAAAG TCTGGAAAGA CTAGTACTGC TACAGATGGT AGAACAGCAT  AF322109 (vif)AACCCTACAC CTTGCAAAGG CTCACGTGGC TTAAATTAAC AGAAGACCAT551                                                600 NC_001463 (vif)GTGTTTCAAG TATTGCCATT GTGGAGAGCC AGGAGAAGTA GTACAACAGA  AF322109 (vif)GTATTTCAAG TAATGCCCTT GTGGAGAGCT CGCAAAGGGA TTACCATAGA601                                                650 NC_001463 (vif)TTTCCCATGG TGCAGGGACA CAACGGGATA CACGCATGCG TGGTCTGTCC  AF322109 (vif)CTTTCCCTGG TGCAGGGACA CAAAAGGATT CCTGGAGCCG TGGACAACGC651                                       691 NC_001463 (vif) AGGAGTGCTGGTTGATGGAA TATCTCTTAG AGGATGAGTG A  AF322109 (vif) AAGAGTGTTG GCAAATAGAGTATCCCTTGG AGGATGAGTG A

TABLE 7 Pileup MSF: 736 Type: N Check: 513 . . . Name: NC_001463(gag720bp) (SEQ ID NO: 15) Len: 736 Check: 4701 Weight: 0 Name:           >AF015181 (SEQ ID NO: 16) Len: 736 Check: 5812 Weight: 0 //1                                                   50 NC_001463(gag720bp) .ATGGTGAGT CTAGATAGAG ACATGGCGAG GCAAGTCTCC GGGGGGAAAA           >AF015181 GCTGTAGACT CTGTAATGTT CCAACAA.AT GCAAA....CAGTAGCAATG 51                                                 100NC_001463 (gag720bp) GAGATTATCC TGAGCTCGAA AAATGTATCA AGCATGCATGCAAGATAAAA            >AF015181 CAGCATGGCC TCGTGTCCGA GGATTTTGAAAGACAGTTAG CAT.ATTATG101                                                150 NC_001463(gag720bp) GTTCGACTCA CAGGGG..AG CACTTGACAG AAGGAAATTG TTTATGGTGC           >AF015181 CTACTACCTG GACAAGTAAA GACATACTAG AAGTA..TTGGCCATGATGC 151                                                200NC_001463 (gag720bp) CTTAAAACA. ...TTAGATT ..ACATGTTT GAGGACCAT..AAAGAGGAA            >AF015181 CTGGGAATAG GGCTCAGAAA GAACTTATTCAAGGGAAATT GAATGAAGAA201                                                250 NC_001463(gag720bp) CCTTGGACAA AAGTAAAATT TAGGACAATA TGGCAGAAGG .TGAAGAATC           >AF015181 GCA..GACAG GTGGAGAAG. ..GAACAATC CACCAGGAGGATTAACAGTG 251                                                300NC_001463 (gag720bp) TAACTCCTGA GGAGAGTAAC AAAAAAGACT TTATGTCTTTGCAGGCCACA            >AF015181 GATCAAATTA TGGGGGTAGG ACAAACAAAT..GCA..... GCAGCACAAG301                                                350 NC_001463(gag720bp) TTAGCGGGTC TAATGTGTTG CCAAATGGGG ATGAGACCTG AGACATTGCA           >AP015181 CTAACATGGA TCAGGCAA.G ACAAATATGC CT...ACAATGGGTAATAAA 351                                                400NC_001463 (gag720bp) AGATGCAATG GCTACAGTAA TCATGAAAGA TGGGTTACTGGAACAAGAGG            >AF015181 CGCCTTAAGA GCAGTAAGGC ATATGGCTCATAGGCCAGGG AATCCAATGC401                                                450 NC_001463(gag720bp) AAAAGAAGGA AGACAAAAGA GAAAAGGAAG AGAGTGTCTT CCCAATAGTA           >AF015181 TAGTAAAGCA A...AAAACA AATGAGCCAT ATGAAGAATTTGCAGCAAGA 451                                                500NC_001463 (gag720bp) GTGCAAGCAG CA..GGAGGG AGAAGCTGGA AAGCAGTAGATTCTGTAATG            >AF015181 CTGCTAGAAG CAATAGATGC AGAAGCGGTTACACAGCCCA TAAAAGAGTA501                                                550 NC_001463(gag720bp) T.TCCAGC.A ACTGCAAACA GTAGCAATGC AGCATGGCCT CGTGTCTGAG           >AF015181 TCTAAAGCTA ACATTATCCT ATACAAATGC AGC.....CTCA........ 551                                                600NC_001463 (gag720bp) GACTTTGAAA GGCAGTTGGC ATATTATGCT ACTACCTGGACAAGTAAAGA            >AF015181 GATTGTCAAA AGCAAATGG. AGAGAGTGCTAGGACAAAGA ...GTACA.A601                                                650 NC_001463(gag720bp) CATACTAGAA GTATTGGCCA TGATGCCTGG AAATAGAGCT CAAAAGGAGT           >AF015181 CAGGCTAGT. GTAGAAAAAA AAATGCAAGC ATGT................ 651                                                700NC_001463 (gag720bp) TAATTCAAGG GAAATTAAAT GAAGAAGCAG AAAGGTGGAGAAGGAATAAT            >AF015181 .......... .......... .................... .......... 701                                   736NC_001463 (gag720bp) CCACCACCTC CAGCAGGAGG AGGATTAACA GTGGAT           >AF015181 .......... .......... .......... ...... Pileup MSF:1347 Type: N Check: 939 . . . Name: NC_001463 (gag) (SEQ ID NO: 17) Len:1347 Check: 6959 Weight: 0 Name:       >AF015181 (SEQ ID NO: 18) Len:1347 Check: 3980 Weight: 0 //1                                                   50 NC_001463 (gag)ATGGTGAGTC TAGATAGAGA CATGGCGAGG CAAGTCTCCG GGGGGAAAAG       >AF015181.......... .......... .......... .......... ..........51                                                 100 NC_001463 (gag)AGATTATCCT GAGCTCGAAA AATGTATCAA GCATGCATGC AAGATAAAAG       >AF015181.......... .......... .......... .......... ..........101                                                150 NC_001463 (gag)TTCGACTCAG AGGGGAGCAC TTGACAGAAG GAAATTGTTT ATGGTGCCTT       >AF015181.......... .......... .......... .......... ..........151                                                200 NC_001463 (gag)AAAACATTAG ATTACATGTT TGAGGACCAT AAAGAGGAAC CTTGGACAAA       >AF015181.......... .......... .......... .......... ..........201                                                250 NC_001463 (gag)AGTAAAATTT AGGACAATAT GGCAGAAGGT GAAGAATCTA ACTCCTGAGG       >AF015181.......... .......... .......... .......... ..........251                                                300 NC_001463 (gag)AGAGTAACAA AAAAGACTTT ATGTCTTTGC AGGCCACATT AGCGGGTCTA       >AF015181.......... .......... .......... .......... ..........301                                                350 NC_001463 (gag)ATGTGTTGCC AAATGGGGAT GAGACCTGAG ACATTGCAAG ATGCAATGGC       >AF015181.......... .......... .......... .......... ..........351                                                400 NC_001463 (gag)TACAGTAATC ATGAAAGATG GGTTACTGGA ACAAGAGGAA AAGAAGGAAG       >AF015181.......... .......... .......... .......... ..........401                                                450 NC_001463(gag)ACAAAAGAGA AAAGGAAGAG AGTGTCTTCC CAATAGTAGT GCAAGCAGCA       >AF015181.......... .......... .......... .......... ..........451                                                500 NC_001463 (gag)GGAGGGAGAA GCTGGAAAGC AGTAGATTCT GTAATGTTCC AGCAACTGCA       >AF015181.......... ........GC TGTAGACTCT GTAATGTTCC AACAAATGCA501                                                550 NC_001463 (gag)AACAGTAGCA ATGCAGCATG GCCTCGTGTC TGAGGACTTT GAAAGGCAGT       >AF015181AACAGTAGCA ATGCAGCATG GCCTCGTGTC CGAGGATTTT GAAAGACAGT551                                                600 NC_001463 (gag)TGGCATATTA TGCTACTACC TGGACAAGTA AAGACATACT AGAAGTATTG       >AF015181TAGCATATTA TGCTACTACC TGGACAAGTA AAGACATACT AGAAGTATTG601                                                650 NC_001463 (gag)GCCATGATGC CTGGAAATAG AGCTCAAAAG GAGTTAATTC AAGGGAAATT       >AF015181GCCATGATGC CTGGGAATAG GGCTCAGAAA GAACTTATTC AAGGGAAATT651                                                700 NC_001463 (gag)AAATGAAGAA GCAGAAAGGT GGAGAAGGAA TAATCCACCA CCTCCAGCAG       >AF015181GAATGAAGAA GCAGACAGGT GGAGAAGGAA CAATCCACCA ..........701                                                750 NC_001463 (gag)GAGGAGGATT AACAGTGGAT CAAATTATGG GGGTAGGACA AACAAATCAA       >AF015181..GGAGGATT AACAGTGGAT CAAATTATGG GGGTAGGACA AACAAATCAA751                                                800 NC_001463 (gag)GCAGCAGCAC AAGCTAACAT GGATCAGGCA AGGCAAATAT GCCTGCAATG       >AF015181GCAGCAGCAC AAGCTAACAT GGATCAGGCA AGACAAATAT GCCTACAATG801                                                850 NC_001463 (gag)GGTAATAAAT GCATTAAGAG CAGTAAGACA TATGGCGCAC AGGCCAGGGA       >AF015181GGTAATAAAC GCCTTAAGAG CAGTAAGGCA TATGGCTCAT AGGCCAGGGA851                                                900 NC_001463 (gag)ATCCAATGCT AGTAAAGCAA AAAACGAATG AGCCATATGA AGATTTTGCA       >AF015181ATCCAATGCT AGTAAAGCAA AAAACAAATG AGCCATATGA AGAATTTGCA901                                                950 NC_001463 (gag)GCAAGACTGC TAGAAGCAAT AGATGCAGAG CCAGTTACAC AGCCTATAAA       >AF015181GCAAGACTGC TAGAAGCAAT AGATGCAGAA GCGGTTACAC AGCCCATAAA951                                               1000 NC_001463 (gag)AGATTATCTA AAGCTAACAC TATCTTATAC AAATGCATCA GCAGATTGTC       >AF015181AGAGTATCTA AAGCTAACAT TATCCTATAC AAATGCAGCC TCAGATTGTC1001                                              1050 NC_001463 (gag)AGAAGCAAAT GGATAGAACA CTAGGACAAA GAGTACAACA AGCTAGTGTA       >AF015181AAAAGCAAAT GGAGAGAGTG CTAGGACAAA GAGTACAACA GGCTAGTGTA1051                                              1100 NC_001463 (gag)GAAGAAAAAA TGCAAGCATG TAGAGATGTG GGATCAGAAG GGTTCAAAAT       >AF015181GAAAAAAAAA TGCAAGCATG T......... .......... ..........1101                                              1150 NC_001463 (gag)GCAATTGTTA GCACAAGCAT TAAGGCCAGG AAAAGGAAAA GGGAATGGAC       >AF015181.......... .......... .......... .......... ..........1151                                              1200 NC_001463 (gag)AGCCACAAAG GTGTTACAAC TGTGGAAAAC CGGGACATCA AGCAAGGCAA       >AF015181.......... .......... .......... .......... ..........1201                                              1250 NC_001463 (gag)TGTAGACAAG GAATCATATG TCACAACTGT GGAAAGAGAG GACATATGCA       >AF015181.......... .......... .......... .......... ..........1251                                              1300 NC_001463 (gag)AAAAGAATGC AGAGGAAAGA GAGACATAAG GGGAAAACAG CAGGGAAACG       >AF015181.......... .......... .......... .......... ..........1301                                           1347 NC_001463 (gag)GGAGGAGGGG GATACGTGTG GTGCCGTCCG CTCCTCCTAT GGAATAA       >AF015181.......... .......... .......... .......... .......

TABLE 8 Pileup MSF: 727 Type: N Check: 1231 . . . Name: NC_001463(gag720bp) (SEQ ID NO: 19) Len: 727 Check: 1714 Weight: 0 Name:            >AF402664 (SEQ ID NO: 20) Len: 727 Check: 1659 Weight: 0Name:             >AF402665 (SEQ ID NO: 21) Len: 727 Check:  331 Weight:0 Name:             >AF402666 (SEQ ID NO: 22) Len: 727 Check: 7190Weight: 0 Name:             >AF402667 (SEQ ID NO: 23) Len: 727 Check:9833 Weight: 0 Name:             >AF402668 (SEQ ID NO: 24) Len: 727Check:  504 Weight: 0 //1                                                   50 NC_001463(gag720bp) ATGGTGAGTC TAGATAGAGA CATGGCGAGG CAAGTCTCCG GGGGGAAAAG           >AF402664 ........TC AAGCAGCAGG .AGGGAGAAG CTGGAAAGCAGTAGACTCAG            >AF402665 ........GC AAGCAGCAGG .AGGGAGAAGCTGGAAAGCA GTAGACTCAG            >AF402666 ........GC AAGCAGCAGG.AGGGAGAAG CTGGAAAGCA GTAGACTCAG            >AF402667 ........GCAAGCAGCAGG .AGGGAGAAG CTGGAAAGCA GTAGACTCAG            >AF402668........GC AAGCAGCAGG .AGGGAGAAG CTGGAAAGCA GTAGACTCAG51                                                 100 NC_001463(gag720bp) AGATTATCCT GAGCTCGAAA AATGTATCAA GCATGCATGC AAGATAAAAG           >AF402664 TGATGTTCCA GCAACTGCAA AATGTAGCAA TGCAGCATGGCCTCGTGTCC            >AF402665 TGATGTTCCA GCAACTGCAA AATGTAGCAATGCAGCATGG CCTCGTGTCC            >AF402666 TGATGTTCCA GCAACTGCAAAATGTAGCAA TGCAGCATGG CCTCGTGTCC            >AF402667 TGATGTTCCAGCAACTGCAA AATGTAGCAA TGCAGCATGG CCTCGTGTCC            >AF402668TGATGTTCCA GCAACTGCAA AATGTAGCAA TGCAGCATGG CCTCGTGTCC101                                                150 NC_001463(gag720bp) TTCGACTCAG AGGGGAGCAC TTGACAGAAG GAAATTGTTT ATGGTGCCTT           >AF402664 GAGGATTTTG AAAGG..CAG TTAGTATATT ATGCTACTACCTGGACAAGT            >AF402665 GAGGATTTTG AAAGG..CAG TTAGCATATTATGCTACTAC CTGGACAAGT            >AF402666 GAGGATTTTG AAAGG..CAGTTGGCATATT ATGCTACTAC CTGGACAAGT            >AF402667 GAGGATTTTGAAAGG..CAG TTAGCATATT ATGCTACTAC CTGGACAAGT            >AF402668GAGGATTTTG AAAGG..CAG TTAGCATATT ATGCTACTAC CTGGACAAGT151                                                200 NC_001463(gag720bp) AAAACATTAG ATTACATGTT TGAGGACCAT AAAGAGGAAC CITGGACAAA           >AF402664 AAAGA..TAT ATTAGAAGTA TTGG..CCAT GATG.....CCTGGAAATAG            >AF402665 AAAGA..TAT ATTAGAAGTA TTGG..CCATGATG.....C CTGGAAATAG            >AF402666 AAAGA..TAT ATTAGAAGTATTGG..CCAT GATG.....C CTGGAAACAG            >AF402667 AAAGA..TATATTAGAAGTA TTGG..CCAT GATG.....C CTGGAAATAG            >AF402668AAAGA..TAT ATTAGAAGTA TTGG..CCAT GATG.....C CTGGAAATAG201                                                250 NC_001463(gag720bp) AGTAAAATTT AGGACAATAT GGCAGAAGGT GAAGAATCTA ACTCCTGAGG           >AF402664 AGCTCAAAAA GAGTTAATTC AAGGGAAATT GAATGAGGAAGCAGAAAGGT            >AF402665 AGCTCAAAAA GAGTTAATTC AAGGGAAATTGAATGAGGAA GCAGAAAGGT            >AF402666 AGCTCAAAAA GAGTTAATTCAGGGGAAATT GAATAAGGAA GCAGAAAGGT            >AF402667 AGCTCAAAAAGAGTTAATTC AAGGGAAATT GAATGAGGAA GCAGAAAGGT            >AF402668AGCTCAAAAA GAGTTAATTC AAGGGAAATT GAATGAGGAA GCAGAAAGGT251                                                300 NC_001463(gag720bp) AGAGTAACAA AAAAGACTTT ATGTCTTTGC AGGCCACATT AGCGGGTCTA           >AF402664 GGAG.AAGAA ATAATCCACC ACCTCAA.GC AGGCG......GAGGATTAA            >AF402665 GGAG.AAGAA ATAATCCACC ACCTCAA.GCAGGCG..... .GAGGATTAA            >AF402666 GGAG.AAGAA ATAATCCACCACCTCAA.GC AGGCG..... .GAGGATTAA            >AF402667 GGAG.AAGAAATAATCCACC ACCTCAA.GC AGGCG..... .GAGGATTAA            >AF402668GGAG.AAGAA ATAATCCACC ACCTCAA.GC AGGCG..... .GAGGATTAA301                                                350NC_001463(gag720bp) ATGTGTTGCC AA..ATGGGG ATGAGACCTG AGACATTGCAAGATGCAATG            >AF4 02664 CAGTGGATCA AATTATGGGG GTAGGACAAACAAATCAAGC AGCGGCACAG            >AF4 02665 CAGTGGATCA AATTATGGGGGTAGGACAAA CAAATCAAGC AGCGGCACAG            >AF4 02666 CAGTGGATCAAATTATGGGG GTAGGACAAA CAAATCAGGC AGCGGCACAG            >AF4 02667CAGTGGATCA AATTATGGGG GTAGGACAAA CAAATCAAGC AGCGGCACAG            >AF402668 CAGTGGATCA AATTATGGGG GTAGGACAAA CAAATCAAGC AGCGGCACAG351                                                400 NC_001463(gag720bp) GCTA.CAGTA ATCATGAAAG ATGGGTT..A CTGGAACAAG .AGGAAAAGA           >AF402664 GCTAACATGG ATCAGGCAAG ACAAATATGT CTGCAATGGGTAATAACAGC            >AF402665 GCTAACATGG ATCAGGCAAG ACAAATATGCCTGCAATGGG TAATAACAGC            >AF402666 GCTAACATGG ATCAGGCAAGACAAATATGC CTGCAATGGG TAATAACAGC            >AF402667 GCTAACATGGATCAGGCAAG ACAAATATGC CTGCAATGGG TAATAACAGC            >AF402668GCTAACATGG ATCAGGCAAG ACAAATATGC CTGCAATGGG TAATAACAGC401                                                450 NC_001463(gag720bp) AGGAAGACAA AAGAGAAAAG GAAGAGAGTG TCTTCCCAAT AGTAGTGCAA           >AF402664 ACTAAGAGCA GTGAGACATA TGGCTCACAA ACCAGGGAATCCAA.TGCTA            >AF402665 ACTAAGAGCA GTGAGACATA TGGCTCACAAACCAGGGAAT CCAA.TGCTA            >AF402666 ACTAAGAGCA GTGAGACATATGGCTCACAA ACCAGGGAAT CCAA.TGCTA            >AF402667 ACTAAGAGCAGTGAGACATA TGGCTCACAA ACCAGGGAAT CCAA.TGCTA            >AF402668ACTAAGAGCA GTGAGACATA TGGCTCACAA ACCAGGGAAT CCAA.TGCTA451                                                500 NC_001463(gag720bp) GCAGCAGGAG GGAGAAGCTG GAAAGCAGTA GATTCTGTAA TCTTCCAGCA           >AF402664 GTAAAGCAAA AAACAAATGA GTCATATGAA GATTTTGCCGCAAGACTGCT            >AF402665 GTAAAGCAAA AGACAAATGA GTCATATGAAGATTTTGCCG CAAGACTGCT            >AF402666 GTAAAGCAAA AGACAAATGAGTCATATGAA GATTTTGCCG CAAGACTGCT            >AF402667 GTAAAGCAAAAGACAAATGA GTCATATGAA GATTTTGCCG CAAGACTGCT            >AF402668GTAAAGCAAA AGACAAATGA GTCATATGAA AAATTTTCAG CAAGACTCCT501                                                550 NC_001463(gag720bp) ACTGCAAACA GTAGCA.ATG CAGCATGGCC TCGTGTCTGA GGACTTTGAA           >AF402664 AGAAGCAATA GATGCAGAAC CAGTTACACA GCAAATAAAAGAATATTTAA            >AF402665 AGAAGCAATA GATGCAGAAC CAGTTACACAGCAAATAAAA GAATATTTAA            >AF402666 AGAAGCAATA GATGCAGAACCAGTTACACA GCAAATAAAA GAATATTTAA            >AF402667 AGAAGCAATAGATGCAGAAC CAGTTACACA GCAAATAAA. GAATATTTAA            >AF402668AGAAGCAATA GATGCAGAAC CAGTTACACA GCCTATAAAA GAATATTTAA551                                                600 NC_001463(gag720bp) AGGCAGTTGG CATATTATGC TACTACCTGG ACAAGTAAAG ACATACTAGA           >AF402664 AGTTA..... .ACATTATCT TAC.ACAAAT GCATCCTCAGACTGTCAGAA            >AF402665 AGTTA..... .ACATTATCT TAC.ACAAATGCATCCTCAG ACTGTCAAAA            >AF402666 AGTTA..... .ACATTATCTTAC.ACAAAT GCATCCTCAG ACTGTCAGAA            >AF402667 .................... .......... .......... ..........            >AF402668AGTTA..... .ACATTATCT TAC.ACAAAT GCATCCTCAG ACTGTCAAAA601                                                650 NC_001463(gag720bp) AGTATTGGCC ATGATGCCTG GAAATAGAGC TCAAAAGGAG TTAATTCAAG           >AF402664 ACAGATGGAT AGAGTACTAG GACAGAGAGT GCAACAAGCTAGTGTGGAAG            >AF402665 ACAAATGGAT AGAATACTAG GACAGAGAGTGCAACAAGCT AGTGTGGAAG            >AF402666 ACAAATGGAT AGAGTACTAGGACAGAGAGT GCAACAAGCT AGTGTGGAAG            >AF402667 .................... .......... .......... ..........            >AF402668ACAAATGGAT AGAGTACTAG GACAGAGAGT GCAACAAGCT AGTGTGGAAG651                                                700 NC_001463(gag720bp) GGAAATTAAA TGAAGAAGCA GAAAGGTGGA GAAGGAATAA TCCACCACCT           >AF402664 AAAAAATGCA ACCAT..GCA GAGATGTGGG ATCAGAAGGATTCAGAATGC            >AF402665 AAAAAATGCA ACCAT..GCA GAGATGTGGGATCAGAAGGG TTCAGAATGC            >AF402666 AAAAAATGCA AGCAT..GCAGAGATGTGGG ATCAGAAGG. ..........            >AF402667 .................... .......... .......... ..........            >AF402668AAAAAATGCA AGCAT..GCA GAGATGTGGG ATCAGAAGGA TTCAGAATGC701                       727 NC_001463 (gag720bp) CCAGCAGGAG GAGGATTAACAGTGGAT            >AF402664 .......... .......... .......           >AF402665 .......... .......... .......            >AF402666.......... .......... .......            >AF402667 .......... .................            >AF402668 .......... .......... ....... Pileup MSF:1347 Type: N Check: 5320 . . . Name: NC_001463 (gag) (SEQ ID NO: 25)Len: 1347 Check: 6959 Weight: 0 Name:       >AF402664 (SEQ ID NO: 26)Len: 1347 Check: 1590 Weight: 0 Name:       >AF402665 (SEQ ID NO: 27)Len: 1347 Check: 9222 Weight: 0 Name:       >AF402666 (SEQ ID NO: 28)Len: 1347 Check: 4950 Weight: 0 Name:       >AF402667 (SEQ ID NO: 29)Len: 1347 Check: 3156 Weight: 0 Name:       >AF402668 (SEQ ID NO: 30)Len: 1347 Check: 9443 Weight: 0 //1                                                   50 NC_001463 (gag)ATGGTGAGTC TAGATAGAGA CATGGCGAGG CAAGTCTCCG GGGGGAAAAG       >AF402664.......... .......... .......... .......... ..........       >AF402665.......... .......... .......... .......... ..........       >AF402666.......... .......... .......... .......... ..........       >AF402667.......... .......... .......... .......... ..........       >AF402668.......... .......... .......... .......... ..........51                                                 100 NC_001463 (gag)AGATTATCCT GAGCTCGAAA AATGTATCAA GCATGCATGC AAGATAAAAG       >AF402664.......... .......... .......... .......... ..........       >AF402665.......... .......... .......... .......... ..........       >AF402666.......... .......... .......... .......... ..........       >AF402667.......... .......... .......... .......... ..........       >AF402668.......... .......... .......... .......... ..........101                                                150 NC_001463(gag)TTCGACTCAG AGGGGAGCAC TTGACAGAAG GAAATTGTTT ATGGTGCCTT       >AF402664.......... .......... .......... .......... ..........       >AF402665.......... .......... .......... .......... ..........       >AF402666.......... .......... .......... .......... ..........       >AF402667.......... .......... .......... .......... ..........       >AF402668.......... .......... .......... .......... ..........151                                                200 NC_001463(gag)AAAACATTAG ATTACATGTT TGAGGACCAT AAAGAGGAAC CTTGGACAAA       >AF402664.......... .......... .......... .......... ..........       >AF402665.......... .......... .......... .......... ..........       >AF402666.......... .......... .......... .......... ..........       >AF402667.......... .......... .......... .......... ..........       >AF402668.......... .......... .......... .......... ..........201                                                250 NC_001463 (gag)AGTAAAATTT AGGACAATAT GGCAGAAGGT GAAGAATCTA ACTCCTGAGG       >AF402664.......... .......... .......... .......... ..........       >AF402665.......... .......... .......... .......... ..........       >AF402666.......... .......... .......... .......... ..........       >AF402667.......... .......... .......... .......... ..........       >AF402668.......... .......... .......... .......... ..........251                                                300 NC_001463 (gag)AGAGTAACAA AAAAGACTTT ATGTCTTTGC AGGCCACATT AGCGGGTCTA       >AF402664.......... .......... .......... .......... ..........       >AF402665.......... .......... .......... .......... ..........       >AF402666.......... .......... .......... .......... ..........       >AF402667.......... .......... .......... .......... ..........       >AF402668.......... .......... .......... .......... ..........301                                                350 NC_001463 (gag)ATGTGTTGCC AAATGGGGAT GAGACCTGAG ACATTGCAAG ATGCAATGGC       >AF402664.......... .......... .......... .......... ..........       >AF402665.......... .......... .......... .......... ..........       >AF402666.......... .......... .......... .......... ..........       >AF402667.......... .......... .......... .......... ..........       >AF402668.......... .......... .......... .......... ..........351                                                400 NC_001463 (gag)TACAGTAATC ATGAAAGATG GGTTACTGGA ACAAGAGGAA AAGAAGGAAG       >AF402664.......... .......... .......... .......... ..........       >AF402665.......... .......... .......... .......... ..........       >AF402666.......... .......... .......... .......... ..........       >AF402667.......... .......... .......... .......... ..........       >AF402668.......... .......... .......... .......... ..........401                                                450 NC_001463 (gag)ACAAAAGAGA AAAGGAAGAG AGTGTCTTCC CAATAGTAGT GCAAGCAGCA       >AF402664.......... .......... .......... .......... TCAAGCAGCA       >AF402665.......... .......... .......... .......... GCAAGCAGCA       >AF402666.......... .......... .......... .......... GCAAGCAGCA       >AF402667.......... .......... .......... .......... GCAAGCAGCA       >AF402668.......... .......... .......... .......... GCAAGCAGCA451                                                500 NC_001463 (gag)GGAGGGAGAA GCTGGAAAGC AGTAGATTCT GTAATGTTCC AGCAACTGCA       >AF402664GGAGGGAGAA GCTGGAAAGC AGTAGACTCA GTGATGTTCC AGCAACTGCA       >AF402665GGAGGGAGAA GCTGGAAAGC AGTAGACTCA GTGATGTTCC AGCAACTGCA       >AF402666GGAGGGAGAA GCTGGAAAGC AGTAGACTCA GTGATGTTCC AGCAACTGCA       >AF402667GGAGGGAGAA GCTGGAAAGC AGTAGACTCA GTGATGTTCC AGCAACTGCA       >AF402668GGAGGGAGAA GCTGGAAAGC AGTAGACTCA GTGATGTTCC AGCAACTGCA501                                                550 NC_001463(gag)AACAGTAGCA ATGCAGCATG GCCTCGTGTC TGAGGACTTT GAAAGGCAGT       >AF402664AAATGTAGCA ATGCAGCATG GCCTCGTGTC CGAGGATTTT GAAAGGCAGT       >AF402665AAATGTAGCA ATGCAGCATG GCCTCGTGTC CGAGGATTTT GAAAGGCAGT       >AF402666AAATGTAGCA ATGCAGCATG GCCTCGTGTC CGAGGATTTT GAAAGGCAGT       >AF402667AAATGTAGCA ATGCAGCATG GCCTCGTGTC CGAGGATTTT GAAAGGCAGT       >AF402668AAATGTAGCA ATGCAGCATG GCCTCGTGTC CGAGGATTTT GAAAGGCAGT551                                                600 NC_001463 (gag)TGGCATATTA TGCTACTACC TGGACAAGTA AAGACATACT AGAAGTATTG       >AF402664TAGTATATTA TGCTACTACC TGGACAAGTA AAGATATATT AGAAGTATTG       >AF402665TAGCATATTA TGCTACTACC TGGACAAGTA AAGATATATT AGAAGTATTG       >AF402666TGGCATATTA TGCTACTACC TGGACAAGTA AAGATATATT AGAAGTATTG       >AF402667TAGCATATTA TGCTACTACC TGGACAAGTA AAGATATATT AGAAGTATTG       >AF402668TAGCATATTA TGCTACTACC TGGACAAGTA AAGATATATT AGAAGTATTG601                                                650 NC_001463 (gag)GCCATGATGC CTGGAAATAG AGCTCAAAAG GAGTTAATTC AAGGGAAATT       >AF402664GCCATGATGC CTGGAAATAG AGCTCAAAAA GAGTTAATTC AAGGGAAATT       >AF402665GCCATGATGC CTGGAAATAG AGCTCAAAAA GAGTTAATTC AAGGGAAATT       >AF402666GCCATGATGC CTGGAAACAG AGCTCAAAAA GAGTTAATTC AGGGGAAATT       >AF402667GCCATGATGC CTGGAAATAG AGCTCAAAAA GAGTTAATTC AAGGGAAATT       >AF402668GCCATGATGC CTGGAAATAG AGCTCAAAAA GAGTTAATTC AAGGGAAATT651                                                700 NC_001463 (gag)AAATGAAGAA GCAGAAAGGT GGAGAAGGAA TAATCCACCA CCTCCAGCAG       >AF402664GAATGAGGAA GCAGAAAGGT GGAGAAGAAA TAATCCACCA CCTCAAGCAG       >AF402665GAATGAGGAA GCAGAAAGGT GGAGAAGGAA TAATCCACCA CCTCAAGCAG       >AF402666GAATAAGGAA GCAGAAAGGT GGAGAAGGAA TAATCCACCA CCTCAAGCAC       >AF402667GAATGAGGAA GCAGAAAGGT GGAGAAGGAA TAATCCACCA CCTCAAGCAG       >AF402668GAATGAGGAA GCAGAAAGGT GGAGAAGGAA TAATCCACCA CCTCAAGCAG701                                                750 NC_001463 (gag)GAGGAGGATT AACAGTGGAT CAAATTATGG GGGTAGGACA AACAAATCAA       >AF402664GCGGAGGATT AACAGTGGAT CAAATTATGG GGGTAGGACA AACAAATCAA       >AF402665GCGGAGGATT AACAGTGGAT CAAATTATGG GGGTAGGACA AACAAATCAA       >AF402666AAGGAGGATT AACAGTGGAT CAAATTATGG GGGTAGGACA AACAAATCAG       >AF402667GCGGAGGATT AACAGTGGAT CAAATTATGG GGGTAGGACA AACAAATCAA       >AF402668GCGGAGGATT AACAGTGGAT CAAATTATGG GGGTAGGACA AACAAATCAA751                                                800 NC_001463 (gag)GCAGCAGCAC AAGCTAACAT GGATCAGGCA AGGCAAATAT GCCTGCAATG       >AF402664GCAGCGGCAC AGGCTAACAT GGATCAGGCA AGACAAATAT GTCTGCAATG       >AF402665GCAGCGGCAC AGGCTAACAT GGATCAGGCA AGACAAATAT GCCTGCAATG       >AF402666GCAGCGGCAC AGGCTAACAT GGATCAGGCA AGACAAATAT GCCTGCAATG       >AF402667GCAGCGGCAC AGGCTAACAT GGATCAGGCA AGACAAATAT GCCTGCAATG       >AF402668GCAGCGGCAC AGGCTAACAT GGATCAGGCA AGACAAATAT GCCTGCAATG801                                                850 NC_001463 (gag)GGTAATAAAT GCATTAAGAG CAGTAAGACA TATGGCGCAC AGGCCAGGGA       >AF402664GGTAATAACA GCACTAAGAG CAGTGAGACA TATGGCTCAC AAACCAGGGA       >AF402665GGTAATAACA GCACTAAGAG CAGTGAGACA TATGGCTCAC AAACCAGGGA       >AF402666GGTAATAACA GCACTAAGAG CAGTGAGACA TATGGCTCAC AAACCAGGGA       >AF402667GGTAATAACA GCACTAAGAG CAGTGAGACA TATGGCTCAC AAACCAGGGA       >AF402668GGTAATAACA GCACTAAGAG CAGTGAGACA TATGGCTCAC AAACCAGGGA852                                                900 NC_001463 (gag)ATCCAATGCT AGTAAAGCAA AAAACGAATG AGCCATATGA AGATTTTGCA       >AF402664ATCCAATGCT AGTAAAGCAA AAAACAAATG AGTCATATGA AGATTTTGCC       >AF402665ATCCAATGCT AGTAAAGCAA AAGACAAATG AGTCATATGA AGATTTTGCC       >AF402666ATCCAATGCT AGTAAAGCAA AAGACAAATG AGTCATATGA AGATTTTGCC       >AF402667ATCCAATGCT AGTAAAGCAA AAGACAAATG AGTCATATGA AGATTTTGCC       >AF402668ATCCAATGCT AGTAAAGCAA AAGACAAATG AGTCATATGA AAAATTTTCA901                                                950 NC_001463 (gag)GCAAGACTGC TAGAAGCAAT AGATGCAGAG CCAGTTACAC AGCCTATAAA       >AF402664GCAAGACTGC TAGAAGCAAT AGATGCAGAA CCAGTTACAC AGCAAATAAA       >AF402665GCAAGACTGC TAGAAGCAAT AGATGCAGAA CCAGTTACAC AGCAAATAAA       >AF402666GCAAGACTGC TAGAAGCAAT AGATGCAGAA CCAGTTACAC AGCAAATAAA       >AF402667GCAAGACTGC TAGAAGCAAT AGATGCAGAA CCAGTTACAC AGCAAATAAA       >AF402668GCAAGACTCC TAGAAGCAAT AGATGCAGAA CCAGTTACAC AGCCTATAAA951                                               1000 NC_001463 (gag)AGATTATCTA AAGCTAACAC TATCTTATAC AAATGCATCA GCAGATTGTC       >AF402664AGAATATTTA AAGTTAACAT TATCTTACAC AAATGCATCC TCAGACTGTC       >AF402665AGAATATTTA AAGTTAACAT TATCTTACAC AAATGCATCC TCAGACTGTC       >AF402666AGAATATTTA AAGTTAACAT TATCTTACAC AAATGCATCC TCAGACTGTC       >AF402667.GAATATTTA A......... .......... .......... ..........       >AF402668AGAATATTTA AAGTTAACAT TATCTTACAC AAATGCATCC TCAGACTGTC1001                                              1050 NC_001463 (gag)AGAAGCAAAT GGATAGAACA CTAGGACAAA GAGTACAACA AGCTAGTGTA       >AF402664AGAAACAGAT GGATAGAGTA CTAGGACAGA GAGTGCAACA AGCTAGTGTG       >AF402665AAAAACAAAT GGATAGAATA CTAGGACAGA GAGTGCAACA AGCTAGTGTG       >AF402666AGAAACAAAT GGATAGAGTA CTAGGACAGA GAGTGCAACA AGCTAGTGTG       >AF402667.......... .......... .......... .......... ..........       >AF402668AAAAACAAAT GGATAGAGTA CTAGGACAGA GAGTGCAACA AGCTAGTGTG1051                                              1100 NC_001463 (gag)GAAGAAAAAA TGCAAGCATG TAGAGATGTG GGATCAGAAG GGTTCAAAAT       >AF402664GAAGAAAAAA TGCAAGCATG CAGAGATGTG GGATCAGAAG GATTCAGAAT       >AF402665GAAGAAAAAA TGCAAGCATG CAGAGATGTG GGATCAGAAG GGTTCAGAAT       >AF402666GAAGAAAAAA TGCAAGCATG CAGAGATGTG GGATCAGAAG G.........       >AF402667.......... .......... .......... .......... ..........       >AF402668GAAGAAAAAA TGCAAGCATG CAGAGATGTG GGATCAGAAG GATTCAGAAT1101                                              1150 NC_001463 (gag)GCAATTGTTA GCACAAGCAT TAAGGCCAGG AAAAGGAAAA GGGAATGGAC       >AF402664GC........ .......... .......... .......... ..........       >AF402665GC........ .......... .......... .......... ..........       >AF402666.......... .......... .......... .......... ..........       >AF402667.......... .......... .......... .......... ..........       >AF402668GC........ .......... .......... .......... ..........1151                                              1200 NC_001463 (gag)AGCCACAAAG GTGTTACAAC TGTGGAAAAC CGGGACATCA AGCAAGGCAA       >AF402664.......... .......... .......... .......... ..........       >AF402665.......... .......... .......... .......... ..........       >AF402666.......... .......... .......... .......... ..........       >AF402667.......... .......... .......... .......... ..........       >AF402668.......... .......... .......... .......... ..........1201                                              1250 NC_001463 (gag)TGTAGACAAG GAATCATATG TCACAACTGT GGAAAGAGAG GACATATGCA       >AF402664.......... .......... .......... .......... ..........       >AF402665.......... .......... .......... .......... ..........       >AF402666.......... .......... .......... .......... ..........       >AF402667.......... .......... .......... .......... ..........       >AF402668.......... .......... .......... .......... ..........1251                                              1300 NC_001463 (gag)AAAAGAATGC AGAGGAAAGA GAGACATAAG GGGAAAACAG CAGGGAAACG       >AF402664.......... .......... .......... .......... ..........       >AF402665.......... .......... .......... .......... ..........       >A~402666.......... .......... .......... .......... ..........       >AF402667.......... .......... .......... .......... ..........       >AF402668.......... .......... .......... .......... ..........1301                                           1347 NC_001463 (gag)GGAGGAGGGG GATACGTGTG GTGCCGTCCG CTCCTCCTAT GGAATAA       >AF402664.......... .......... .......... .......... .......       >AF402665.......... .......... .......... .......... .......       >AF402666.......... .......... .......... .......... .......       >AF402667.......... .......... .......... .......... .......       >AF402668.......... .......... .......... .......... .......

TABLE 9 Pileup MSF: 742 Type: N Check: 6523 . . . Name: NC_001463(gag720bp) (SEQ ID NO: 31) Len: 742 Check: 3818 Weight: 0 Name:           >AJ305040 (SEQ ID NO: 32) Len: 742 Check: 1263 Weight: 0Name:            >AJ305041 (SEQ ID NO: 33) Len: 742 Check: 9126 Weight:0 Name:            >AJ305042 (SEQ ID NO: 34) Len: 742 Check: 2316Weight: 0 // 1                                                   50NC_001463 (gag720bp) ATGGTGAGTC TAGATAGAGA CATGGCGAGG CAAGTCTC..CGGGGGGAAA            >AJ305040 ......GCAG TCGATGCTGT AATGTTCCAGCAAATGCAAA CAGTAGCCAT            >AJ305041 ......GCAG TAGACTCAGTAATGTTCCAG CAACTGCAAA CAGTAGCAAT            >AJ305042 ......GCAGTCGATGCTGT AATGTTCCAG CAAATGCAAA CAGTAGCCAT51                                                 100 NC_001463(gag720bp) AGAGATTATC CTGAGCTCGA AAAATGTATC AAGCATGCAT GCAAGATAAA           >AJ305040 GCAGCATGGT CTTGTGTCTG AGGACTTTGA AAGGCAGTTAGCAT.ATTGT            >AJ305041 GCAGCATGGC CTCGTGTCCG AGGATTTTGAAAGGCAGTTG GCAT.ATTAT            >AJ305042 GCAGCATGGT CTTGTGTCTGAGGACTTTGA AAGGCAGTTA GCAT.ATTAT101                                                150 NC_001463(gag720bp) AGTTCGACTC AGAGGGG..A GCACTTGACA GAAGGAAATT GTTTATGGTG           >AJ305040 GCTACTACCT GGACAAGTAA AGATATAITA GAAGTA..TTGGCCATGATG            >AJ305041 GCTACTACCT GGACGAGTAA AGACATACTAGAAGTA..TT GGCCATGATG            >AJ305042 GCTACTACCT GGACAAGTAAAGATATATTA GAAGTA..TT GGCCATGATG151                                                200 NC_001463(gag720bp) CCTTAAAACA TTAGATTACA TGTTTGAGGA CCATAAAGAG GAACCTTGGA           >AJ305040 CCTGGAAATA G.AGCTCAAA AA...GAGTT AATTCAAG.GAAAATTAAAC            >AJ305041 CCTGGAAACA G.AGCTCAAA AG...GAGTTAATTCAAG.G GAAATTAAAT            >AJ305042 CCTGGAAATA G.AGCTCAAAAA...GAGTT AATTCAAG.G AAAATTAAAT201                                                250 NC_001463(gag720bp) CAAAAGTAAA ATTTAGGACA ATATGGCAGA AGGTGAAGAA TCTAACTCCT           >AJ305040 GAGGAAGCAG AA..AGGTGG AGAAGGAATA A..TCCACCGCCTCCACAAG            >AJ305041 GAAGAGGCAG AA..AGGTGG AGAAGACATAA..TCCACCC CCTCCGGCGG            >AJ305042 GAGGAAGCAG AA..AGGTGGAGAAGGAATA A..TCCACCG CCTCCACAGG251                                                300 NC_001463(gag720bp) GAGGAG.AGT AACAAAAAAG .ACTTTATGT CTTTGCAGGC CACATTAGCG           >AJ305040 GAGGGGGATT AACAGTGGAT CAAATTATGG GGAT..AGGACAAACAAATC            >AJ305041 GAGGAGGATT AACAGTGGAT CAAATTATGGGGGT..AGGA CAAACAAATC            >AJ305042 GAGGGGGATT AACAGTGGATCAAATTATGG GGAT..AGGA CAAACAAATC301                                                350 NC_001463(gag720bp) GGTCTAATGT GTTGCCAAAT GGGGATGAGA CCTGAGACAT .....TGCAA           >AJ305040 AAGCAGCAGC ACAAGCTAAC ATGGATCAGG CAAGACACATATGCCTGCAA            >AJ305041 AAGCAGCAGC ACAAGCTAAC ATGGATCAGGCAAGACAAAT ATGCCTGCAA            >AJ305042 AAGCAGCAGC ACAAGCTAACATGGATCAGG CAAGACACAT ATGCCTGCAA351                                                400 NC_001463(gag720bp) GATGCAATGG CTACAGTAAT ..CA.TGAAA GATGGGTTAC TGGAACAAGA           >AJ305040 TGGGTAATAA CAGCATTAAG AGCAGTAAGA CATATGGCTCACAGACCAGG            >AJ305041 TGGGTAATAA CAGCATTAAG AGCAGTGAGGTATATGACTC ACAAACCAGG            >AJ305042 TGGGTAATAA CAGCATTAAGAGCAGTAAGA CATATGGCTC ACAGACCAGG401                                                450 NC_001463(gag720bp) GGA...AAAG A.AGGAAGAC AAAAGAGAAA AGGAAGAGAG T..GTCTTCC           >AJ305040 GAATCCAATG CTCGTAAAAC AAAAAACAAA TGAGCCATATGAAGAGTTTG            >AJ305041 GAATCCAATG CTAGTAAAAC AAAAAACAAATGAAGCATAT GAAGAGTTTA            >AJ305042 GAATCCAATG CTCGTAAAACAAAAAACAAA TGAGCCATAT GAAGAGTTTG451                                                500 NC_001463(gag720bp) CAATAGTAGT GCAAGCAGCA GGAG..GGAG AAGCTGGAAA GCAGTAGATT           >AJ305040 CAGCAAAACT ATTAGAAGCA ATAGATGCAG AACCAGTAACACAGCCCATA            >AJ305041 CAGCGAGACT GCTAGAAGCA ATAGATGCAGAGCCAGTAAC ACAGCCCACA            >AJ305042 CAGCAAAACT ATTAGAAGCAATAGATGCAG AACCAGTAAC ACAGCTCATA501                                                550 NC_001463(gag720bp) CTGTAATGTT CCAGCAACTG CAAACAGTAG CAATGCAGCA TGGCCTCGTG           >AJ305040 AAAGACTAT.. CTAAAGTT.. .AACATTAT CT.TATACAAATGCGTC...            >AJ305041 AAAGAATAT.. CTAAAACT.. .AACATTATCT.TATACAA ATGCATC...            >AJ305042 AAAGACTAT.. CTAAAGTT...AACATTAT CT.TATACAA ATGCGTC...551                                                600 NC_001463(gag720bp) TCTGAGGACT TTGAAAGGCA GTTGGCATAT TATGCTACTA CCTGGACAAG           >AJ305040 .CTCAG.ACT GTCAAAAGCA AATGG.ATAG AGTGCTGGGACAAAG...AG            >AJ305041 .CTCAG.ACT GTCAAAAGCA AATGG.ATAGAGTACTAGGA CAAAG...AG            >AJ305042 .CTCAG.ACT GTCAAAAGCAAATGG.ATAG AGTGCTGGGA CAAAG...AG601                                                650 NC_001463(gag720bp) TAAAGACATA CTAGAAGTAT TGGCCATGAT GCCTGGAAAT AGAGCTCAAA           >AJ305040 TGCA.ACAAG CTAGT.GTAG ACGAGAAAAT GCAA................            >AJ305041 TGCA.ACAAG CTAGT.GTAG AAGAAAAAATGCAA...... ..........            >AJ305042 TGCA.ACAAG CTAGT.GTAGACGAGAAGAT GCAA...... ..........651                                                700 NC_001463(gag720bp) AGGAGTTAAT TCAAGGGAAA TTAAATGAAG AAGCAGAAAG GTGGAGAAGG           >AJ305040 .......... .......... .......... ....................            >AJ305041 .......... .......... .................... ..........            >AJ305042 .......... .................... .......... ..........701                                        742 NC_001463 (gag72obp)AATAATCCAC CACCTCCAGC AGGAGGAGGA TTAACAGTGG AT            >AJ305040.......... .......... .......... .......... ..            >AJ305041.......... .......... .......... .......... ..            >AJ305042.......... .......... .......... .......... .. PileUp MSF: 1347 Type: NCheck: 9510 . . . Name: NC_001463 (gag) (SEQ ID NO: 35) Len: 1347 Check:6959 Weight: 0 Name:       >AJ305040 (SEQ ID NO: 36) Len: 1347 Check:1930 Weight: 0 Name:       >AJ305041 (SEQ ID NO: 37) Len: 1347 Check:7682 Weight: 0 Name:       >AJ305042 (SEQ ID NO: 38) Len: 1347 Check:2939 Weight: 0 // 1                                                   50NC_001463 (gag) ATGGTGAGTC TAGATAGAGA CATGGCGAGG CAAGTCTCCG GGGGGAAAAG      >AJ305040 .......... .......... .......... .......... ..........      >AJ305041 .......... .......... .......... .......... ..........      >AJ305042 .......... .......... .......... .......... ..........51                                                 100 NC_001463 (gag)AGATTATCCT GAGCTCGAAA AATGTATCAA GCATGCATGC AAGATAAAAG       >AJ305040.......... .......... .......... .......... ..........       >AJ305041.......... .......... .......... .......... ..........       >AJ305042.......... .......... .......... .......... ..........101                                                150 NC_001463 (gag)TTCGACTCAG AGGGGAGCAC TTGACAGAAG GAAATTGTTT ATGGTGCCTT       >AJ305040.......... .......... .......... .......... ..........       >AJ305041.......... .......... .......... .......... ..........       >AJ305042.......... .......... .......... .......... ..........151                                                200 NC_001463 (gag)AAAACATTAG ATTACATGTT TGAGGACCAT AAAGAGGAAC CTTGGACAAA       >AJ305040.......... .......... .......... .......... ..........       >AJ305041.......... .......... .......... .......... ..........       >AJ305042.......... .......... .......... .......... ..........201                                                250 NC_001463 (gag)AGTAAAATTT AGGACAATAT GGCAGAAGGT GAAGAATCTA ACTCCTGAGG       >AJ305040.......... .......... .......... .......... ..........       >AJ305041.......... .......... .......... .......... ..........       >AJ305042.......... .......... .......... .......... ..........251                                                300 NC_001463 (gag)AGAGTAACAA AAAAGACTTT ATGTCTTTGC AGGCCACATT AGCGGGTCTA       >AJ305040.......... .......... .......... .......... ..........       >AJ305041.......... .......... .......... .......... ..........       >AJ305042.......... .......... .......... .......... ..........301                                                350 NC_001463 (gag)ATGTGTTGCC AAATGGGGAT GAGACCTGAG ACATTGCAAG ATGCAATGGC       >AJ305040.......... .......... .......... .......... ..........       >AJ305041.......... .......... .......... .......... ..........       >AJ305042.......... .......... .......... .......... ..........351                                                400 NC_001463 (gag)TACAGTAATC ATGAAAGATG GGTTACTGGA ACAAGAGGAA AAGAAGGAAG       >AJ305040.......... .......... .......... .......... ..........       >AJ305041.......... .......... .......... .......... ..........       >AJ305042.......... .......... .......... .......... ..........401                                                450 NC_001463 (gag)ACAAAAGAGA AAAGGAAGAG AGTGTCTTCC CAATAGTAGT GCAAGCAGCA       >AJ305040.......... .......... .......... .......... ..........       >AJ305041.......... .......... .......... .......... ..........       >AJ305042.......... .......... .......... .......... ..........451                                                500 NC_001463 (gag)GGAGGGAGAA GCTGGAAAGC AGTAGATTCT GTAATGTTCC AGCAACTGCA       >AJ305040.......... ........GC AGTCGATGCT GTAATGTTCC AGCAAATGCA       >AJ305041.......... ........GC AGTAGACTCA GTAATGTTCC AGCAACTGCA       >AJ305042.......... ........GC AGTCGATGCT GTAATGTTCC AGCAAATGCA501                                                550 NC_001463 (gag)AACAGTAGCA ATGCAGCATG GCCTCGTGTC TGAGGACTTT GAAAGGCAGT       >AJ305040AACAGTAGCC ATGCAGCATG GTCTTGTGTC TGAGGACTTT GAAAGGCAGT       >AJ305041AACAGTAGCA ATGCAGCATG GCCTCGTGTC CGAGGATTTT GAAAGGCAGT       >AJ305042AACAGTAGCC ATGCAGCATG GTCTTGTGTC TGAGGACTTT GAAAGGCAGT551                                                600 NC_001463 (gag)TGGCATATTA TGCTACTACC TGGACAAGTA AAGACATACT AGAAGTATTG       >AJ305040TAGCATATTG TGCTACTACC TGGACAAGTA AAGATATATT AGAAGTATTG       >AJ305041TGGCATATTA TGCTACTACC TGGACGAGTA AAGACATACT AGAAGTATTG       >AJ305042TAGCATATTA TGCTACTACC TGGACAAGTA AAGATATATT AGAAGTATTG601                                                650 NC_001463 (gag)GCCATGATGC CTGGAAATAG AGCTCAAAAG GAGTTAATTC AAGGGAAATT       >AJ305040GCCATGATGC CTGGAAATAG AGCTCAAAAA GAGTTAATTC AAGGAAAATT       >AJ305041GCCATGATGC CTGGAAACAG AGCTCAAAAG GAGTTAATTC AAGGGAAATT       >AJ305042GCCATGATGC CTGGAAATAG AGCTCAAAAA GAGTTAATTC AAGGAAAATT651                                                700 NC_001463 (gag)AAATGAAGAA GCAGAAAGGT GGAGAAGGAA TAATCCACCA CCTCCAGCAG       >AJ305040AAACGAGGAA GCAGAAAGGT GGAGAAGGAA TAATCCACCG CCTCCACAAG       >AJ305041AAATGAAGAG GCAGAAAGGT GGAGAAGACA TAATCCACCC CCTCCGGCGG       >AJ305042AAATGAGGAA GCAGAAAGGT GGAGAAGGAA TAATCCACCG CCTCCACAGG701                                                750 NC_001463 (gag)GAGGAGGATT AACAGTGGAT CAAATTATGG GGGTAGGACA AACAAATCAA       >AJ305040GAGGGGGATT AACAGTGGAT CAAATTATGG GGATAGGACA AACAAATCAA       >AJ305041GAGGAGGATT AACAGTGGAT CAAATTATGG GGGTAGGACA AACAAATCAA       >AJ305042GAGGGGGATT AACAGTGGAT CAAATTATGG GGATAGGACA AACAAATCAA751                                                800 NC_001463 (gag)GCAGCAGCAC AAGCTAACAT GGATCAGGCA AGGCAAATAT GCCTGCAATG       >AJ305040GCAGCAGCAC AAGCTAACAT GGATCAGGCA AGACACATAT GCCTGCAATG       >AJ305041GCAGCAGCAC AAGCTAACAT GGATCAGGCA AGACAAATAT GCCTGCAATG       >AJ305042GCAGCAGCAC AAGCTAACAT GGATCAGGCA AGACACATAT GCCTGCAATG801                                                850 NC_001463(gag)GGTAATAAAT GCATTAAGAG CAGTAAGACA TATGGCGCAC AGGCCAGGGA       >AJ305040GGTAATAACA GCATTAAGAG CAGTAAGACA TATGGCTCAC AGACCAGGGA       >AJ305041GGTAATAACA GCATTAAGAG CAGTGAGGTA TATGACTCAC AAACCAGGGA       >AJ305042GGTAATAACA GCATTAAGAG CAGTAAGACA TATGGCTCAC AGACCAGGGA851                                                900 NC_001463 (gag)ATCCAATGCT AGTAAAGCAA AAAACGAATG AGCCATATGA AGATTTTGCA       >AJ305040ATCCAATGCT CGTAAAACAA AAAACAAATG AGCCATATGA AGAGTTTGCA       >AJ305041ATCCAATGCT AGTAAAACAA AAAACAAATG AAGCATATGA AGAGTTTACA       >AJ305042ATCCAATGCT CGTAAAACAA AAAACAAATG AGCCATATGA AGAGTTTGCA901                                                950 NC_001463 (gag)GCAAGACTGC TAGAAGCAAT AGATGCAGAG CCAGTTACAC AGCCTATAAA       >AJ305040GCAAAACTAT TAGAAGCAAT AGATGCAGAA CCAGTAACAC AGCCCATAAA       >AJ305041GCGAGACTGC TAGAAGCAAT AGATGCAGAG CCAGTAACAC AGCCCACAAA       >AJ305042GCAAAACTAT TAGAAGCAAT AGATGCAGAA CCAGTAACAC AGCTCATAAA951                                               1000 NC_001463 (gag)AGATTATCTA AAGCTAACAC TATCTTATAC AAATGCATCA GCAGATTGTC       >AJ305040AGACTATCTA AAGTTAACAT TATCTTATAC AAATGCGTCC TCAGACTGTC       >AJ305041AGAATATCTA AAACTAACAT TATCTTATAC AAATGCATCC TCAGACTGTC       >AJ305042AGACTATCTA AAGTTAACAT TATCTTATAC AAATGCGTCC TCAGACTGTC1001                                              1050 NC_001463 (gag)AGAAGCAAAT GGATAGAACA CTAGGACAAA GAGTACAACA AGCTAGTGTA       >AJ305040AAAAGCAAAT GGATAGAGTG CTGGGACAAA GAGTGCAACA AGCTAGTGTA       >AJ305041AAAAGCAAAT GGATAGAGTA CTAGGACAAA GAGTGCAACA AGCTAGTGTA       >AJ305042AAAAGCAAAT GGATAGAGTG CTGGGACAAA GAGTGCAACA AGCTAGTGTA1051                                              1100 NC_001463 (gag)GAAGAAAAAA TGCAAGCATG TAGAGATGTG GGATCAGAAG GGTTCAAAAT       >AJ305040GACGAGAAAA TGCAA..... .......... .......... ..........       >AJ305041GAAGAAAAAA TGCAA..... .......... .......... ..........       >AJ305042GACGAGAAGA TGCAA..... .......... .......... ..........1101                                              1150 NC_001463 (gag)GCAATTGTTA GCACAAGCAT TAAGGCCAGG AAAAGGAAAA GGGAATGGAC       >AJ305040.......... .......... .......... .......... ..........       >AJ305041.......... .......... .......... .......... ..........       >AJ305042.......... .......... .......... .......... ..........1151                                              1200 NC_001463 (gag)AGCCACAAAG GTGTTACAAC TGTGGAAAAC CGGGACATCA AGCAAGGCAA       >AJ305040.......... .......... .......... .......... ..........       >AJ305041.......... .......... .......... .......... ..........       >AJ305042.......... .......... .......... .......... ..........1201                                              1250 NC_001463 (gag)TGTAGACAAG GAATCATATG TCACAACTGT GGAAAGAGAG GACATATGCA       >AJ305040.......... .......... .......... .......... ..........       >AJ305041.......... .......... .......... .......... ..........       >AJ305042.......... .......... .......... .......... ..........1251                                              1300 NC_001463 (gag)AAAAGAATGC AGAGGAAAGA GAGACATAAG GGGAAAACAG CAGGGAAACG       >AJ305040.......... .......... .......... .......... ..........       >AJ305041.......... .......... .......... .......... ..........       >AJ305042.......... .......... .......... .......... ..........1301                                           1347 NC_001463 (gag)GGAGGAGGGG GATACGTGTG GTGCCGTCCG CTCCTCCTAT GGAATAA       >AJ305040.......... .......... .......... .......... ..........       >AJ305041.......... .......... .......... .......... ..........       >AJ305042.......... .......... .......... .......... ..........

TABLE 10 Pileup MSF: 728 Type: N Check: 9403 . . . Name:NC_001463(gag720bp) (SEQ ID NO: 39) Len: 728 Check: 5765 Weight: 0 Name:          >AY047362 (SEQ ID NO: 40) Len: 728 Check: 3638 Weight: 0 //1                                                   50 NC_001463(gag720bp) ATGGTGAGTC TAGATAGAGA CATGGCGAGG CAAGTCTCCG GGGGGAAAAG          >AY047362 .......... .......... .......... .................... 51                                                 100NC_001463 (gag720bp) AGATTATCCT GAGCTCGAAA AATGTATCAA GCATGCATGCAAGATAAAAG           >AY047362 ......TAAA GATATATTAG AA.GTATTGGCCATG.ATGC CTGGAAATAG101                                                150 NC_001463(gag720bp) TTCGACTCAG AGGGGAGCAC TTGACAGAAG GAAATTGTTT ATGGTGCCTT          >AY047362 AGC...TCAA AAAGAGTTAA TTCA...AGG GAAATTGAATGAAGAAGCAG 151                                                200NC_001463 (gag720bp) AAAACATTAG ATTACATGTT TGAGGACCAT AAAGAGGAACCTTGGACAAA           >AY047362 AAAGGTGGAG AAGGAATAAT CCACCACCTCAAGCAGG..C GGAGGATTAA201                                                250 NC_001463(gag720bp) AGTAAAATTT AGGACAATAT GGCAGAAGGT GAAG..AATC TAACTCCTGA          >AY047362 C..AG..TGG ATCAAATTAT GGGGGTAGGA CAAACAAATCAAGCAGCGGC 251                                                300NC_001463 (gag720bp) GGAGAGTAAC AAAAAAGACT TTATGTCTTT GCAGGCCACATTAGCGGGTC           >AY047362 ACAGGCTAAC ATGGATCAG. ..........GCAAGACAAA TATGC..CTG301                                                350 NC_001463(gag720bp) TAATGTGTTG CCAAATGGGG ATGAGACCTG AGACATTGCA AGATGCAATG          >AY047362 CAATGGGTAA TAACAGCACT AAGAGCAGTG AGACAT......A.....TG 351                                                400NC_001463 (gag720bp) GCTACAGTAA TCATGAAAGA TGGGTTACTG GAACAAGAGGAAAAGAAGGA           >AY047362 GCT.CACAAA CCAGGGA..A TCCGATGCT......ACT.. AAAGCAA..A401                                                450 NC_001463(gag720bp) AGACAAAAGA GAAA.AGGAA GAGAGTGTCT TCCCAATAGT AGTGCAAGCA          >AY047362 AAACAAATGA GTCATATGAA GATTTTGCCG ...CAAGACTGCTAGAAGCA 451                                                500NC_001463 (gag720bp) GCAGGAGGGA GAAGCTGGAA AGCAGTAGAT TCTGTAATGTTCCAGCAACT           >AY047362 ATAG.ATGCA GAACCAGTTA CAAAGCAAATAAAAGAATAT TT.....AAA501                                                550 NC_001463(gag720bp) GCAAACAGTA GCAATGCAGC ATGGCCTCGT GTCTGAGGAC TTTGAAAGGC          >AY047362 GTTAACATTA TCT.TACACA AATGCATC.. ..CTCAG.ACTGTAAGAAAC 551                                                600NC_001463 (gag720bp) AGTTGGCATA TTATGCTACT ACCTGGACAA GTA.AAGACATACTAGAAGT           >AY047362 AGATGG.ATA GAGTACTAGG ACAGAGAGTGCAACAAGCTA GTGTGGAAGA601                                                650 NC_001463(gag720bp) ATTG..GCCA TGATGCCTGG AAATAGAGCT CAAAAGGAGT TA..ATTCAA          >AY047362 AAAAATGCAA GCATGCAGAG ATGT.GGGAT CAGAAGGATTCAGAATGC.. 651                                                700NC_001463 (gag720bp) GGGAAATTAA ATGAAGAAGC AGAAAGGTGG AGAAGGAATAATCCACCACC           >AY047362 .......... .......... .................... .......... 701                        728 NC_001463(gag720bp)TCCAGCAGGA GGAGGATTAA CAGTGGAT           >AY047362 .......... .................. Pileup MSF: 1347 Type: N Check: 3238 . . . Name: NC_001463(gag) (SEQ ID NO: 41) Len: 1347 Check: 6959 Weight: 0 Name:      >AY047362 (SEQ ID NO: 42) Len: 1347 Check: 6279 Weight: 0 //1                                                   50 NC_001463 (gag)ATGGTGAGTC TAGATAGAGA CATGGCGAGG CAAGTCTCCG GGGGGAAAAG       >AY047362.......... .......... .......... .......... ..........51                                                 100 NC_001463 (gag)AGATTATCCT GAGCTCGAAA AATGTATCAA GCATGCATGC AAGATAAAAG       >AY047362.......... .......... .......... .......... ..........101                                                150 NC_001463 (gag)TTCGACTCAG AGGGGAGCAC TTGACAGAAG GAAATTGTTT ATGGTGCCTT       >AY047362.......... .......... .......... .......... ..........151                                                200 NC_001463 (gag)AAAACATTAG ATTACATGTT TGAGGACCAT AAAGAGGAAC CTTGGACAAA       >AY047362.......... .......... .......... .......... ..........201                                                250 NC_001463 (gag)AGTAAAATTT AGGACAATAT GGCAGAAGGT GAAGAATCTA ACTCCTGAGG       >AY047362.......... .......... .......... .......... ..........251                                                300 NC_001463 (gag)AGAGTAACAA AAAAGACTTT ATGTCTTTGC AGGCCACATT AGCGGGTCTA       >AY047362.......... .......... .......... .......... ..........301                                                350 NC_001463 (gag)ATGTGTTGCC AAATGGGGAT GAGACCTGAG ACATTGCAAG ATGCAATGGC       >AY047362.......... .......... .......... .......... ..........351                                                400 NC_001463 (gag)TACAGTAATC ATGAAAGATG GGTTACTGGA ACAAGAGGAA AAGAAGGAAG       >AY047362.......... .......... .......... .......... ..........401                                                450 NC_001463 (gag)ACAAAAGAGA AAAGGAAGAG AGTGTCTTCC CAATAGTAGT GCAAGCAGCA       >AY047362.......... .......... .......... .......... ..........451                                                500 NC_001463 (gag)GGAGGGAGAA GCTGGAAAGC AGTAGATTCT GTAATGTTCC AGCAACTGCA       >AY047362.......... .......... .......... .......... ..........501                                                550 NC_001463 (gag)AACAGTAGCA ATGCAGCATG GCCTCGTGTC TGAGGACTTT GAAAGGCAGT       >AY047362.......... .......... .......... .......... ..........551                                                600 NC_001463 (gag)TGGCATATTA TGCTACTACC TGGACAAGTA AAGACATACT AGAAGTATTG       >AY047362.......... .......... ........TA AAGATATATT AGAAGTATTG601                                                650 NC_001463 (gag)GCCATGATGC CTGGAAATAG AGCTCAAAAG GAGTTAATTC AAGGGAAATT       >AY047362GCCATGATGC CTGGAAATAG AGCTCAAAAA GAGTTAATTC AAGGGAAATT651                                                700 NC_001463 (gag)AAATGAAGAA GCAGAAAGGT GGAGAAGGAA TAATCCACCA CCTCCAGCAG       >AY047362GAATGAAGAA GCAGAAAGGT GGAGAAGGAA TAATCCACCA CCTCAAGCAG701                                                750 NC_001463 (gag)GAGGAGGATT AACAGTGGAT CAAATTATGG GGGTAGGACA AACAAATCAA       >AY047362GCGGAGGATT AACAGTGGAT CAAATTATGG GGGTAGGACA AACAAATCAA751                                                800 NC_001463 (gag)GCAGCAGCAC AAGCTAACAT GGATCAGGCA AGGCAAATAT GCCTGCAATG       >AY047362GCAGCGGCAC AGGCTAACAT GGATCAGGCA AGACAAATAT GCCTGCAATG801                                                850 NC_001463 (gag)GGTAATAAAT GCATTAAGAG CAGTAAGACA TATGGCGCAC AGGCCAGGGA       >AY047362GGTAATAACA GCACTAAGAG CAGTGAGACA TATGGCTCAC AAACCAGGGA851                                                900 NC_001463 (gag)ATCCAATGCT AGTAAAGCAA AAAACGAATG AGCCATATGA AGATTTTGCA       >AY047362ATCCGATGCT AGTAAAGCAA AAAACAAATG AGTCATATCA AGATTTTGCC901                                                950 NC_001463 (gag)GCAAGACTGC TAGAAGCAAT AGATGCAGAG CCAGTTACAC AGCCTATAAA       >AY047362GCAAGACTGC TAGAAGCAAT AGATGCAGAA CCAGTTACAA AGCAAATAAA951                                               1000 NC_001463 (gag)AGATTATCTA AAGCTAACAC TATCTTATAC AAATGCATCA GCAGATTGTC       >AY047362AGAATATTTA AAGTTAACAT TATCTTACAC AAATGCATCC TCAGACTGTA1001                                              1050 NC_001463 (gag)AGAAGCAAAT GGATAGAACA CTAGGACAAA GAGTACAACA AGCTAGTGTA       >AY047362AGAAACAGAT GGATAGAGTA CTAGGACAGA GAGTGCAACA AGCTAGTGTG1051                                              1100 NC_001463 (gag)GAAGAAAAAA TGCAAGCATG TAGAGATGTG GGATCAGAAG GGTTCAAAAT       >AY047362GAAGAAAAAA TGCAAGCATG CAGAGATGTG GGATCAGAAG GATTCAGAAT1101                                              1150 NC_001463 (gag)GCAATTGTTA GCACAAGCAT TAAGGCCAGG AAAAGGAAAA GGGAATGGAC       >AY047362GC........ .......... .......... .......... ..........1151                                              1200 NC_001463 (gag)AGCCACAAAG GTGTTACAAC TGTGGAAAAC CGGGACATCA AGCAAGGCAA       >AY047362.......... .......... .......... .......... ..........1201                                              1250 NC_001463 (gag)TGTAGACAAG GAATCATATG TCACAACTGT GGAAAGAGAG GACATATGCA       >AY047362.......... .......... .......... .......... ..........1251                                              1300 NC_001463 (gag)AAAAGAATGC AGAGGAAAGA GAGACATAAG GGGAAAACAG CAGGGAAACG       >AY047362.......... .......... .......... .......... ..........1301                                           1347 NC_001463 (gag)GGAGGAGGGG GATACGTGTG CTGCCGTCCG CTCCTCCTAT GGAATAA       >AY047362.......... .......... .......... .......... ..........

TABLE 11 Pileup MSF: 733 Type: N Check: 5855 . . . Name: NC_001463(gag720bp) (SEQ ID NO: 43) Len: 733 Check: 9482 Weight: 0 Name:           >AY081139 (SEQ ID NO: 44) Len: 733 Check: 6373 Weight: 0 //1                                                   50 NC_001463(gag720bp) ATGGTGAGTC TAGATAGAGA CATGGCGAGG CAAGTCTCCG GGGGGAAAAG           >AY081139 .......... .......... .......... ......TGCCGTAGACTCTG 51                                                 100NC_001463 (gag720bp) AGATTATCCT G.AGCTCGAA AAATGTATCA AGCATGCATGCAAGATAAAA            >AY081139 TGATGTTCCA CCAGCT.GCA TACAGTAGCAATGCCGCATG GCCTCGTGTC101                                                150 NC_001463(gag720bp) GTTCGACTCA GAGGGGAGCA CTTGACAGAA GGAAATTGTT TATGGTGCCT           >AY081139 TGAGGACTTT GAAAGG..CA GTTGGCATAT TATGCTACTACCTGGACAAG 151                                                200NC_001463 (gag720bp) TAAAACATTA GATTACATGT .TTGAGGACC ATAAAGAGGAACCTTGGACA            >AY081139 TAAAGA..TA TACTGGAAGT ATTGGCCATGATGCCTGGGA ATAGAGCTCA201                                                250 NC_001463(gag720bp) AAAGTAA..A ATTTAGGACA ATATGGCAGA AGGTGAAGAA TCTAACTCCT           >AY081139 AAAAGAATTA ATTCAAGGAA AATTAAATGA AGAAGCAGAA.......... 251                                                300NC_001463 (gag720bp) GAGGAGAGTA ACAAAAAAGA CTTTATGTCT TTGCAGGCCACATTAGCGGG            >AY081139 .AGGTGGAGA AGGAATAATC CACCACCTCAA.GCAGGCG. .....GAGGA301                                                350 NC_001463(gag720bp) TCTAATGTGT TGCCAA..AT GGGGATGAGA CCTGAGACAT TGCAAGATGC           >AY081139 TTAACAGTGG ATCAAATTAT GGGGGTAGGA CAAACAAATCAAGCAGCTGC 351                                                400NC_001463 (gag720bp) AATGGCTA.C AGTAATCATG AAAGATGGGT TACTGGAACAAGAGGAAAAG            >AY081139 ACAAGCTAAC ATGGATCAGG CAAGACAAATA..TGCCTGC AATGGGTAAT401                                                450 NC_001463(gag720bp) AAGGAAGACA AAAGAGAAAA GGAAGAGAGT GTCTTCCCAA TAGTAGTGCA           >AY081139 ATC..AGCCT TAAGAGCAGT GAGACATA.T GTCT..CATAAACCAGGG.A 451                                                500NC_001463 (gag720bp) AGCAGCAGGA GGGAGAAGCT GGAAAGCAGT AGATTCTGTAATGTTCCAGC            >AY081139 ATCCGCTGCT AGTA.AAGCA AAAAACAAATGAGTCATATG AAGATTTTGC501                                                550 NC_001463(gag720bp) AACTGCAAAC ..AGTAGCAA TGCAGCATGG CCTCGTGTCT GAGGACTTTG           >AY081139 AGCTAGACTG CTAGAAGCAA TAGATCCAGC CCCAGTAGCACATC.CTATA 551                                                600NC_001463 (gag720bp) AAAGGCAGTT GGCATATTAT GCTAC....T ACCTGGACAAGTAAAGACAT            >AY081139 AAAGATTATT TAAAGTTAAC ACTATCTTATACGAATGCAT CATCAGATTG601                                                650 NC_001463(gag720bp) ACTAGAAGTA TTGGCCATGA TGCCTGGAAA TAGAGCTCAA AAGGAGTTAA           >AY081139 TCAAAAGCAA ATGGGTAGAA TGCTAGGATC GAGAGTCCATCA..AGCCAG 651                                                700NC_001463 (gag720bp) TTCAAGGGAA ATTAAATGAA GAAGCAGAAA GGTGGAGAAGGAATAATCCA            >AY081139 TGTGGGCCAA AAAA...... .................... .......... 701                               733NC_001463(gag720bp) CCACCTCCAG CAGGAGGAGG ATTAACAGTG GAT           >AY081139 .......... .......... .......... ... PileUp MSF:1347 Type: N Check: 2072 . . . Name: NC_001463 (gag) (SEQ ID NO: 45)Len: 1347 Check: 6959 Weight: 0 Name:       >AY081139 (SEQ ID NO: 46)Len: 1347 Check: 5113 Weight: 0 //1                                                   50 NC_001463 (gag)ATGGTGAGTC TAGATAGAGA CATGGCGAGG CAAGTCTCCG GGGGGAAAAG       >AY081139.......... .......... .......... .......... ..........51                                                 100 NC_001463 (gag)AGATTATCCT GAGCTCGAAA AATGTATCAA GCATGCATGC AAGATAAAAG       >AY081139.......... .......... .......... .......... ..........101                                                150 NC_001463 (gag)TTCGACTCAG AGGGGAGCAC TTGACAGAAG GAAATTGTTT ATGGTGCCTT       >AY081139.......... .......... .......... .......... ..........151                                                200 NC_001463 (gag)AAAACATTAG ATTACATGTT TGAGGACCAT AAAGAGGAAC CTTGGACAAA       >AY081139.......... .......... .......... .......... ..........201                                                250 NC_001463 (gag)AGTAAAATTT AGGACAATAT GGCAGAAGGT GAAGAATCTA ACTCCTGAGG       >AY081139.......... .......... .......... .......... ..........251                                                300 NC_001463 (gag)AGAGTAACAA AAAAGACTTT ATGTCTTTGC AGGCCACATT AGCGGGTCTA       >AY081139.......... .......... .......... .......... ..........301                                                350 NC_001463 (gag)ATGTGTTGCC AAATGGGGAT GAGACCTGAG ACATTGCAAG ATGCAATGGC       >AY081139.......... .......... .......... .......... ..........351                                                400 NC_001463 (gag)TACAGTAATC ATGAAAGATG GGTTACTGGA ACAAGAGGAA AAGAAGGAAG       >AY081139.......... .......... .......... .......... ..........401                                                450 NC_001463 (gag)ACAAAAGAGA AAAGGAAGAG AGTGTCTTCC CAATAGTAGT GCAAGCAGCA       >AY081139.......... .......... .......... .......... ..........451                                                500 NC_001463 (gag)GGAGGGAGAA GCTGGAAAGC AGTAGATTCT GTAATGTTCC AGCAACTGCA       >AY081139.......... .......TGC CGTAGACTCT GTGATGTTCC ACCAGCTGCA501                                                550 NC_001463 (gag)AACAGTAGCA ATGCAGCATG GCCTCGTGTC TGAGGACTTT GAAAGGCAGT       >AY081139TACAGTAGCA ATGCCGCATG GCCTCGTGTC TGAGGACTTT GAAAGGCAGT551                                                600 NC_001463 (gag)TGGCATATTA TGCTACTACC TGGACAAGTA AAGACATACT AGAAGTATTG       >AY081139TGGCATATTA TGCTACTACC TGGACAAGTA AAGATATACT GGAAGTATTG601                                                650 NC_001463 (gag)GCCATGATGC CTGGAAATAG AGCTCAAAAG GAGTTAATTC AAGGGAAATT       >AY081139GCCATGATGC CTGGGAATAG AGCTCAAAAA GAATTAATTC AAGGAAAATT651                                                700 NC_001463 (gag)AAATGAAGAA GCAGAAAGGT GGAGAAGGAA TAATCCACCA CCTCCAGCAG       >AY081139AAATGAAGAA GCAGAAAGGT GGAGAAGGAA TAATCCACCA CCTCAAGCAG701                                                750 NC_001463 (gag)GAGGAGGATT AACAGTGGAT CAAATTATGG GGGTAGGACA AACAAATCAA       >AY081139GCGGAGGATT AACAGTGGAT CAAATTATGG GGGTAGGACA AACAAATCAA751                                                800 NC_001463 (gag)GCAGCAGCAC AAGCTAACAT GGATCAGGCA AGGCAAATAT GCCTGCAATG       >AY081139GCAGCTGCAC AAGCTAACAT GGATCAGGCA AGACAAATAT GCCTGCAATG801                                                850 NC_001463 (gag)GGTAATAAAT GCATTAAGAG CAGTAAGACA TATGGCGCAC AGGCCAGGGA       >AY081139GGTAATATCA GCCTTAAGAG CAGTGAGACA TATGTCTCAT AAACCAGGGA851                                                900 NC_001463 (gag)ATCCAATGCT AGTAAAGCAA AAAACGAATG AGCCATATGA AGATTTTGCA       >AY081139ATCCGCTGCT AGTAAAGCAA AAAACAAATG AGTCATATGA AGATTTTGCA901                                                950 NC_001463 (gag)GCAAGACTGC TAGAAGCAAT AGATGCAGAG CCAGTTACAC AGCCTATAAA       >AY081139GCTAGACTGC TAGAAGCAAT AGATCCAGCC CCAGTAGCAC ATCCTATAAA951                                               1000 NC_001463 (gag)AGATTATCTA AAGCTAACAC TATCTTATAC AAATGCATCA GCAGATTGTC       >AY081139AGATTATTTA AAGTTAACAC TATCTTATAC GAATGCATCA TCAGATTGTC1001                                              1050 NC_001463 (gag)AGAAGCAAAT GGATAGAACA CTAGGACAAA GAGTACAACA AGCTAGTGTA       >AY081139AAAAGCAAAT GGGTAGAATG CTAGGATCGA GAGTCCATCA AGCCAGTGTG1051                                              1100 NC_001463 (gag)GAAGAAAAAA TGCAAGCATG TAGAGATGTG GGATCAGAAG GGTTCAPAAT       >AY081139GGCCAAAAAA .......... .......... .......... ..........1101                                              1150 NC_001463 (gag)GCAATTGTTA GCACAAGCAT TAAGGCCAGG AAAAGGAAAA GGGAATGGAC       >AY081139.......... .......... .......... .......... ..........1151                                              1200 NC_001463 (gag)AGCCACAAAG GTGTTACAAC TGTGGAAAAC CGGGACATCA AGCAAGGCAA       >AY081139.......... .......... .......... .......... ..........1201                                              1250 NC_001463 (gag)TGTAGACAAG GAATCATATG TCACAACTGT GGAAAGAGAG GACATATGCA       >AY081139.......... .......... .......... .......... ..........1251                                              1300 NC_001463 (gag)AAAAGAATGC AGAGGAAAGA GAGACATAAG GGGAAAACAG CAGGGAAACG       >AY081139.......... .......... .......... .......... ..........1301                                           1347 NC_001463 (gag)GGAGGAGGGG GATACGTGTG GTGCCGTCCG CTCCTCCTAT GGAATAA       >AY081139.......... .......... .......... .......... .......

TABLE 12 Pileup MSF: 731 Type: N Check: 9546 . . . Name: NC_001463(gag720bp) (SEQ ID NO: 47) Len: 731 Check: 7595 Weight: 0 Name:           >AY101347 (SEQ ID NO: 48) Len: 731 Check: 7962 Weight: 0Name: >AY101348 (SEQ ID NO: 49) Len: 731 Check: 3989 Weight: 0 //1                                                   50 NC_001463(gag720bp) ATGGTGAGTC TAGATAGAGA CATGGCGAGG CAAGTCTCCG GGGGGAAAAG           >AY101347 .......... .......... .......... ....................            >AY101348 .......... .......... .................... ..........51                                                 100 NC_001463(gag720bp) AGATTATCCT GAGCTCGAAA AATGTATCAA GCATGCATGC AAGATAAAAG           >AY101347 .......... .......... .....AGCAG TAGATTCTGTAATGTTCCAG            >AY101348 .......... .......... .....AGCCGTAGATTCTGT AATGTTCCAG101                                                150 NC_001463(gag720bp) TTCGACT.CA GAGGGGAGCA CTTGACAGAA GGAAATTGTT TATGGTGCCT           >AY101347 ..CAACTGCA AACAGTAGCA AT..GCAGCA TGGACTCGTGTATGAAGACT            >AY101348 ..CAGCTGCA AACAGTAGCA AT..GCAGCATGGCCTCGTG TCAGAGGACT151                                                200 NC_001463(gag720bp) TAAAACATTA GATTACATGT TTGAGGACCA TAAAGAGGAA CCTTGGACAA           >AY101347 TTGAAAGGCT GTCGGCATAT TATGCTACTA CCTGGAC.AAGTAAAGATAT            >AY101348 TTGAAAGGCT TCCAGCATAT CATGCTACTACCTGGGC.AA GTAAAGATAT201                                                250 NC_001463(gag720bp) AAGTAAAATT TAGGACAATA TGGCAGAAGG TGAAGAATCT AACTCCTGAG           >AY101347 ACTGGAAGTA TTGGCCATGA TGCCTG..G. ....GAATAGAGCTCAAAAA            >AY101348 CTTAGAAGTA CTGGCCATGA TGCCTG..G.....AAATAG AGCTCAAAAA251                                                300 NC_001463(gag720bp) GAGAGTAA.. CAAAAAAGAC TTTATGTCTT TGCAGGCCAC ATTAGCGGGT           >AY101347 GA.ATTAATT CAAGGAAAAT TAAATGAAGA AGCAGAAAGGTGGAGAAGGA            >AY101348 GA.GTTAATT CAAGGGAAAT TAAATGAAGAAGCAGAGAGG TGGAGAAGGA301                                                350 NC_001463(gag720bp) CTAATGTGTT GCCAAATGGG GATGAGACCT GAGACATTGC AAGATGCAAT           >AY101347 ATAATCCACC ACCTCAAGCA GGCG.GAGGA TTAACAGTGGATCAAATTAT            >AY101348 ATAATCCACC ACCTCCAGCA GGAG.GAGGGTTAACAGTGG ATCAAATTAT351                                                400 NC_001463(gag720bp) GGCTACAGTA ATCATGAAAG ATGG.GTTAC TGGAACAAGA GGAAAAGAAG           >AY101347 GGGGGTAGGA CAAACAAATC AAGCAGCTGC ACAAGCTAACATGGATCAGG            >AY101348 GGGAGTAGGA CAAACAAATC AGGCAGCGGCACAAGCAAAC ATGGATCAGG401                                                450 NC_001463(gag720bp) GAAGACAAAA GAGAAAAGGA AGAG.AGTGT CTTCCC.AAT AGTAGTGCAA           >AY101347 CAAGACAAAT ATGCCTGCAA TGGGTAATAT CAGCCTTAAGAGCAGTGAGA            >AY101348 CAAGACAAAT ATGCCTACAA TGGGTGATATCAGCACTAAG AGCAGTAAGG451                                                500 NC_001463(gag720bp) GCAGCAGGAG GGAGAAGCTG GAAAGCAGTA GATTCTGTAA TGTTCCAGCA           >AY101347 .CATATGTCT CATAAACCAG GGAATCCGCT GCTAGTA.AAGCAAAAAACA            >AY101348 .CATATGGCT CACAAGCCAG GGAATCCAATGTTAGTA.AA GCAAAAAGCA501                                                550 NC_001463(gag720bp) ACTG...CAA ACAGTAGCAA TGCAGCATGG CCTCGTGTCT GAGGACTTTG           >AY101347 AATGAGTCAT ATGAAGATTT TGCAGCAAGA CTGCTAGAAGCAATAGATGC            >AY101348 AATGAGCCAT ATGAAGAATT TGCAGCAAGGCTGCTGGAAG CAATAGATGC551                                                600 NC_001463(gag720bp) AAAGGCAGTT GG.CATATTA TGCTACTACC TGGACAAGTA AAGAC.ATAC           >AY101347 AGAGCCAGTA GCACATCCTA TAAAAGAATA CTTA.AAGTTAACACTATCT            >AY101348 CGAGCCAGTT AATCAGCCCA TAAAAGAATATCTA.AAACT AACGTTGTCT601                                                650 NC_001463(gag720bp) TAGAAGTATT GGCCATGATG CCTGGAAATA GAGCTCAAAA GGAGTTAATT           >AY101347 TATACGAATG CATCATCA.G ATTGTCAAAA C....CAAATGGATAGAATG            >AY101348 TATACGAATG CATCCTCA.C ATTGTCAGAAG....CAAAT GGATAGAACA651                                                700 NC_001463(gag720bp) CAAGGGAAAT TAAATCAAGA AGCAGAAAGG TGGAGAAGGA ATAATCCACC           >AY101347 CTGG...AAT CAAGAGTACA ACAAGCTAG. TGTAGAACAAAAAA......            >AY101348 CTAG...GAC AAAGAGTCAA ACAAGCTAG.TGTAGAACAA AAAA...... 701                             731 NC_001463(gag720bp) ACCTCCAGCA GGACGAGGAT TAACAGTGCA T            >AY101347.......... .......... .......... .            >AY101348 .................... .......... . PileUp MSF: 1347 Type: N Check: 2815 . . . Name:NC_001463 (gag) (SEQ ID NO: 50) Len: 1347 Check: 6959 Weight: 0 Name:      >AY101347 (SEQ ID NO: 51) Len: 1347 Check: 969 Weight: 0 Name:      >AY101348 (SEQ ID NO: 52) Len: 1347 Check: 4887 Weight: 0 //1                                                   50 NC_001463 (gag)ATGGTGAGTC TAGATAGAGA CATGGCGAGG CAAGTCTCCG GGGGGAAAAG       >AY101347.......... .......... .......... .......... ..........       >AY101348.......... .......... .......... .......... ..........51                                                 100 NC_001463 (gag)AGATTATCCT GAGCTCGAAA AATGTATCAA GCATGCATGC AAGATAAAAG       >AY101347.......... .......... .......... .......... ..........       >AY101348.......... .......... .......... .......... ..........101                                                150 NC_001463 (gag)TTCGACTCAG AGGGGAGCAC TTGACAGAAG GAAATTGTTT ATGGTGCCTT       >AY101347.......... .......... .......... .......... ..........       >AY101348.......... .......... .......... .......... ..........151                                                200 NC_001463 (gag)AAAACATTAG ATTACATGTT TGAGGACCAT AAAGAGGAAC CTTGGACAAA       >AY101347.......... .......... .......... .......... ..........       >AY101348.......... .......... .......... .......... ..........201                                                250 NC_001463 (gag)AGTAAAATTT AGGACAATAT GGCAGAAGGT GAAGAATCTA ACTCCTGAGG       >AY101347.......... .......... .......... .......... ..........       >AY101348.......... .......... .......... .......... ..........251                                                300 NC_001463 (gag)AGAGTAACAA AAAAGACTTT ATGTCTTTGC AGGCCACATT ACCGGGTCTA       >AY101347.......... .......... .......... .......... ..........       >AY101348.......... .......... .......... .......... ..........301                                                350 NC_001463 (gag)ATGTGTTGCC AAATGGGGAT GAGACCTGAG ACATTGCAAG ATGCAATGGC       >AY101347.......... .......... .......... .......... ..........       >AY101348.......... .......... .......... .......... ..........351                                                400 NC_001463 (gag)TACAGTAATC ATGAAAGATG GGTTACTGGA ACAAGAGGAA AAGAAGGAAG       >AY101347.......... .......... .......... .......... ..........       >AY101348.......... .......... .......... .......... ..........401                                                450 NC_001463 (gag)ACAAAAGAGA AAAGGAAGAG AGTGTCTTCC CAATAGTAGT GCAAGCAGCA       >AY101347.......... .......... .......... .......... ..........       >AY101348.......... .......... .......... .......... ..........451                                                500 NC_001463 (gag)GGAGGGAGAA GCTGGAAAGC AGTAGATTCT GTAATGTTCC AGCAACTGCA       >AY101347.......... .......AGC AGTAGATTCT GTAATGTTCC AGCAACTGCA       >AY101348.......... .......AGC CGTAGATTCT GTAATGTTCC AGCAGCTGCA501                                                550 NC_001463 (gag)AACAGTAGCA ATGCAGCATG GCCTCGTGTC TGAGGACTTT GAAAGGCAGT       >AY101347AACAGTAGCA ATGCAGCATG GACTCGTGTA TGAAGACTTT GAAAGGCTGT       >AY101348AACAGTAGCA ATGCAGCATG GCCTCGTGTC AGAGGACTTT GAAAGGCTTC551                                                600 NC_001463 (gag)TGGCATATTA TGCTACTACC TGGACAAGTA AAGACATACT AGAAGTATTG       >AY101347CGGCATATTA TGCTACTACC TGGACAAGTA AAGATATACT GGAAGTATTG       >AY101348CAGCATATCA TGCTACTACC TGGGCAAGTA AAGATATCTT AGAAGTACTG601                                                650 NC_001463 (gag)GCCATGATGC CTGGAAATAG AGCTCAAAAG GAGTTAATTC AAGGGAAATT       >AY101347GCCATGATGC CTGGGAATAG AGCTCAAAAA GAATTAATTC AAGGAAAATT       >AY101348GCCATGATGC CTGGAAATAG AGCTCAAAAA GAGTTAATTC AAGGGAAATT651                                                700 NC_001463 (gag)AAATGAAGAA GCAGAAAGGT GGAGAAGGAA TAATCCACCA CCTCCAGCAG       >AY101347AAATGAAGAA GCAGAAAGGT GGAGAAGGAA TAATCCACCA CCTCAAGCAG       >AY101348AAATGAAGAA GCAGAGAGGT GGAGAAGGAA TAATCCACCA CCTCCAGCAG701                                                750 NC_001463 (gag)GAGGAGGATT AACAGTGGAT CAAATTATGG GGGTAGGACA AACAAATCAA       >AY101347GCGGAGGATT AACAGTGGAT CAAATTATGG GGGTAGGACA AACAAATCAA       >AY101348GAGGAGGGTT AACAGTGGAT CAAATTATGG GAGTAGGACA AACAAATCAG751                                                800 NC_001463 (gag)GCAGCAGCAC AAGCTAACAT GGATCAGGCA AGGCAAATAT GCCTGCAATG       >AY101347GCAGCTGCAC AAGCTAACAT GGATCAGGCA AGACAAATAT GCCTGCAATG       >AY101348GCAGCGGCAC AAGCAAACAT GGATCAGGCA AGACAAATAT GCCTACAATG801                                                850 NC_001463 (gag)GGTAATAAAT GCATTAAGAG CAGTAAGACA TATGGCGCAC AGGCCAGGGA       >AY101347GGTAATATCA GCCTTAAGAG CAGTGAGACA TATGTCTCAT AAACCAGGGA       >AY101348GGTGATATCA GCACTAAGAG CAGTAAGGCA TATGGCTCAC AAGCCAGGGA851                                                900 NC_001463 (gag)ATCCAATGCT AGTAAAGCAA AAAACGAATG AGCCATATGA AGATTTTGCA       >AY101347ATCCGCTGCT AGTAAAGCAA AAAACAAATG AGTCATATGA AGATTTTGCA       >AY101348ATCCAATGTT AGTAAAGCAA AAAGCAAATG AGCCATATGA AGAATTTGCA901                                                950 NC_001463 (gag)GCAAGACTGC TAGAAGCAAT AGATGCAGAG CCAGTTACAC AGCCTATAAA       >AY101347GCAAGACTGC TAGAAGCAAT AGATGCAGAG CCAGTAGCAC ATCCTATAAA       >AY101348GCAAGGCTGC TGGAAGCAAT AGATGCCGAG CCAGTTAATC AGCCCATAAA951                                               1000 NC_001463 (gag)AGATTATCTA AAGCTAACAC TATCTTATAC AAATGCATCA GCAGATTGTC       >AY101347AGAATACTTA AAGTTAACAC TATCTTATAC GAATGCATCA TCAGATTGTC       >AY101348AGAATATCTA AAACTAACGT TGTCTTATAC GAATGCATCC TCAGATTGTC1001                                              1050 NC_001463 (gag)AGAAGCAAAT GGATAGAACA CTAGGACAAA GAGTACAACA AGCTAGTGTA       >AY101347AAAAGCAAAT GGATAGAATG CTGGAATCAA GAGTACAACA AGCTAGTGTA       >AY101348AGAAGCAAAT GGATAGAACA CTAGGACAAA GAGTCAAACA AGCTAGTGTA1051                                              1100 NC_001463 (gag)GAAGAAAAAA TGCAAGCATG TAGAGATGTG GGATCAGAAG GGTTCAAAAT       >AY101347GAACAAAAAA .......... .......... .......... ..........       >AY101348GAACAAAAAA .......... .......... .......... ..........1101                                              1150 NC_001463 (gag)GCAATTGTTA GCACAAGCAT TAAGGCCAGG AAAAGGAAAA GGGAATGGAC       >AY101347.......... .......... .......... .......... ..........       >AY101348.......... .......... .......... .......... ..........1151                                              1200 NC_001463 (gag)AGCCACAAAG GTGTTACAAC TGTGGAAAAC CGGGACATCA AGCAAGGCAA       >AY101347.......... .......... .......... .......... ..........       >AY101348.......... .......... .......... .......... ..........1201                                              1250 NC_001463 (gag)TGTAGACAAG GAATCATATG TCACAACTGT GGAAAGAGAG GACATATGCA       >AY101347.......... .......... .......... .......... ..........       >AY101348.......... .......... .......... .......... ..........1251                                              1300 NC_001463 (gag)AAAAGAATGC AGAGGAAAGA GAGACATAAG GGGAAAACAG CAGGGAAACG       >AY101347.......... .......... .......... .......... ..........       >AY101348.......... .......... .......... .......... ..........1301                                           1347 NC_001463 (gag)GGAGGAGGGG GATACGTGTG GTGCCGTCCG CTCCTCCTAT GGAATAA       >AY101347.......... .......... .......... .......... .......       >AY101348.......... .......... .......... .......... .......

TABLE 13 Pileup MSF: 720 Type: N Check: 3690 . . . Name: NC_001463(gag720bp) (SEQ ID NO: 53) Len: 720 Check: 5792 Weight: 0 Name:             >L78446 (SEQ ID NO: 54) Len: 720 Check:  272 Weight: 0Name:              >L78447 (SEQ ID NO: 55) Len: 720 Check: 1999 Weight:0 Name:              >L78450 (SEQ ID NO: 56) Len: 720 Check: 9633Weight: 0 Name:              >L78451 (SEQ ID NO: 57) Len: 720 Check:5177 Weight: 0 Name:              >L78453 (SEQ ID NO: 58) Len: 720Check:  817 Weight: 0 //1                                                   50 NC_001463(gag720bp) ATGGTGAGTC TAGATAGAGA CATGGCGAGG CAAGTCTCCG GGGGGAAAAG             >L78446 .......... .......... .......... ....................              >L78447 .......... .......... .................... ..........              >L78450 .......... .................... .......... ..........              >L78451 .................... .......... .......... ..........              >L78453.......... .......... .......... .......... ..........51                                                 100 NC_001463(gag720bp) AGATTATCCT GAGCTCGAAA AATGTATCAA GCATGCATGC AAGATAAAAG             >L78446 .......... .......... .......... ....................              >L78447 .......... .......... .................... ..........              >L78450 .......... .................... .......... ..........              >L78451 .................... .......... .......... ..........              >L78453.......... .......... .......... .......... ..........101                                                150 NC_001463(gag720bp) TTCGACTCAG AGGGGAGCAC TTGACAGAAG GAAATTGTTT ATGGTGCCTT             >L78446 .......... .......... .......... ....................              >L78447 .......... .......... .................... ..........              >L78450 .......... .................... .......... ..........              >L78451 .................... .......... .......... ..........              >L78453.......... .......... .......... .......... ..........151                                                200 NC_001463(gag720bp) AAAACATTAG ATTACATGTT TGAGGACCAT AAAGAGGAAC CTTGGACAAA             >L78446 .......... .......... .......... ....................              >L78447 .......... .......... .................... ..........              >L78450 .......... .................... .......... ..........              >L78451 .................... .......... .......... ..........              >L78453.......... .......... .......... .......... ..........201                                                250 NC_001463(gag720bp) AGTAAAATTT AGGACAATAT GGCAGAAGGT GAAGAATCTA ACTCCTGAGG             >L78446 .......... .......... .......... ....................              >L78447 .......... .......... .................... ..........              >L78450 .......... .................... .......... ..........              >L78451 .................... .......... .......... ..........              >L78453.......... .......... .......... .......... ..........251                                                300 NC_001463(gag720bp) AGAGTAACAA AAAAGACTFF ATGTCTTTGC AGGCCACA~~ AGCGGGTCTA             >L78446 .......... .......... .......... ....................              >L78447 .......... .......... .................... ..........              >L78450 .......... .................... .......... ..........              >L78451 .................... .......... .......... ..........              >L78453.......... .......... .......... .......... ..........301                                                350 NC_001463(gag720bp) ATGTGITGCC AAATGGGGAT GAGACCTGAG ACATTGCAAG ATGCAATGGC             >L78446 .......... .......... .......... ....................              >L78447 .......... .......... .................... ..........              >L78450 .......... .................... .......... ..........              >L78451 .................... .......... .......... ..........              >L78453.......... .......... .......... .......... ..........351                                                400 NC_001463(gag720bp) TACAGTAATC ATGAAAGATG GGTTACTGGA ACAAGAGGAA AAGAAGGAAG             >L78446 .......... .......... .......... ....................              >L78447 .......... .......... .................... ..........              >L78450 .......... .................... .......... ..........              >L78451 .................... .......... .......... ..........              >L78453.......... .......... .......... .......... ..........401                                                450 NC_001463(gag720bp) ACAAAAGAGA AAAGOAAGAG AGTGTCTTCC CAATAGTAGT GCAAGCAGCA             >L78446 .......... .......... .......... ....................              >L78447 .......... .......... .................... ..........              >L78450 .......... .................... .......... ..........              >L78451 .................... .......... .......... ..........              >L78453.......... .......... .......... .......... ..........451                                                500 NC_001463(gag720bp) GGAGOGAGAA GCTGGAAAGC AGTAGATTCT GTAATGTTCC AGCAACTGCA             >L78446 .......... .......... .......... ....................              >L78447 .......... .......... .................... ..........              >L78450 .......... .................... .......... ..........              >L78451 .................... .......... .......... ..........              >L78453.......... .......... .......... .......... ..........501                                                550 NC_001463(gag720bp) AACAGTAGCA ATGCAGCATG GCCTCGTGTC TGAGGACTTT GAAAGGCAGT             >L78446 .......... ...CAGCATG GCCTCGTGTC CGAGGACTTTGAAAGGCAGT              >L78447 .......... ...CAGCATG GAATAGTATCAGAAGAGTTA GAGAGGCAAC              >L78450 .......... ...CAACATGGGATAGTATC AGAGGAATTT GAGAGACAAA              >L78451 .............CAGCATG GACTAGTATC AGAAGAATTT GAAAGGCAGC              >L78453.......... ...CAGCATG GACTTGTGTC CGAAGATTTT GAGAGGCAAT551                                                600 NC_001463(gag720bp) TGGCATATTA TGCTACTACC TGGACAAGTA AAGACATACT AGAAGTATTG             >L78446 TGGCATATTA TGCTACTACC TGGACAAGTA AGGACATATTAGAAGTATTG              >L78447 TGTCTTATTA TGCTACCACT TGGACAAGCAAGGATATCTT AGAGGTACTA              >L78450 TGTCTTATTA TGCTACCACATGOACAAGTA AGGATATTTT AGAAGTACTA              >L78451 TAGCATACTATGCCACAACG TGGACAAGCA AAGACATACT AGAGGTGTTA              >L78453TGGCATATTA TGCTACAACC TGGACTAGTG AAGATATATT AGAAGTATTG601                                                650 NC_001463(gag720bp) GCCATGATGC CTGGAAATAG AGCTCAAAAG GAGTTAATTC AAGGGAAATT             >L78446 GCCATOATGC CAGGAAATAG AGCTCAAAAG GAGCTAATTCAA........              >L78447 GCCATGATGC CTGGCAATAG AGCATTAAAAGAGCTAATAC AA........              >L78450 GCAATGATGC CCGGGAACAGAGCATTAAAG GAGCTGATAC AA........              >L78451 GCCATGATGCCAGGGAATAG AGCACAAAAA GAACTAATAC AA........              >L78453GCTATGATGC CTGGGAATAG AGCACAGAAA GAATTAATAC AA........651                                                700 NC_001463(gag720bp) AAATGAAGAA GCAGAAAGGT GGAGAAGGAA TAATCCACCA CCTCCAGCAG             >L78446 .......... .......... .......... ....................              >L78447 .......... .......... .................... ..........              >L78450 .......... .................... .......... ..........              >L78451 .................... .......... .......... ..........              >L78453.......... .......... .......... .......... ..........701                  720 NC_001463 (gag720bp) GAGGAGGATT AACAGTGGAT             >L78446 .......... ..........              >L78447.......... ..........              >L78450 .......... ..........             >L78451 .......... ..........              >L78453.......... .......... PileUp MSF: 1347 Type: N Check: 6947 . . . Name:NC_001463 (gag) (SEQ ID NO: 59) Len: 1347 Check: 6959 Weight: 0 Name:        >L78446 (SEQ ID NO: 60) Len: 1347 Check: 6690 Weight: 0 Name:        >L78447 (SEQ ID NO: 61) Len:1347 Check: 8417 Weight: 0 Name:        >L78450 (SEQ ID NO: 62) Len: 1347 Check: 6051 Weight: 0 Name:        >L78451 (SEQ ID NO: 63) Len: 1347 Check: 1595 Weight: 0 Name:        >L78453 (SEQ ID NO: 64) Len: 1347 Check: 7235 Weight: 0 //1                                                   50 NC_001463 (gag)ATGGTGAGTC TAGATAGAGA CATGGCGAGG CAAGTCTCCG GGGGGAAAAG         >L78446.......... .......... .......... .......... ..........         >L78447.......... .......... .......... .......... ..........         >L78450.......... .......... .......... .......... ..........         >L78451.......... .......... .......... .......... ..........         >L78453.......... .......... .......... .......... ..........51                                                 100 NC_001463 (gag)AGATTATCCT GAGCTCGAAA AATGTATCAA GCATGCATGC AAGATAAAAG         >L78446.......... .......... .......... .......... ..........         >L78447.......... .......... .......... .......... ..........         >L78450.......... .......... .......... .......... ..........         >L78451.......... .......... .......... .......... ..........         >L78453.......... .......... .......... .......... ..........101                                                150 NC_001463 (gag)TTCGACTCAG AGGGGACCAC TTGACAGAAG GAAATTGTTT ATGGTGCCTT         >L78446.......... .......... .......... .......... ..........         >L78447.......... .......... .......... .......... ..........         >L78450.......... .......... .......... .......... ..........         >L78451.......... .......... .......... .......... ..........         >L78453.......... .......... .......... .......... ..........151                                                200 NC_001463 (gag)AAAACATTAG ATTACATGTT TGAGGACCAT AAAGAGGAAC CTTGGACAAA         >L78446.......... .......... .......... .......... ..........         >L78447.......... .......... .......... .......... ..........         >L78450.......... .......... .......... .......... ..........         >L78451.......... .......... .......... .......... ..........         >L78453.......... .......... .......... .......... ..........201                                                250 NC_001463 (gag)AGTAAAATTT AGGACAATAT GGCAGAAGGT GAAGAATCTA ACTCCTGAGG         >L78446.......... .......... .......... .......... ..........         >L78447.......... .......... .......... .......... ..........         >L78450.......... .......... .......... .......... ..........         >L78451.......... .......... .......... .......... ..........         >L78453.......... .......... .......... .......... ..........251                                                300 NC_001463 (gag)AGAGTAACAA AAAAGACTTT ATGTCTTTGC AGGCCACATT AGCGGGTCTA         >L78446.......... .......... .......... .......... ..........         >L78447.......... .......... .......... .......... ..........         >L78450.......... .......... .......... .......... ..........         >L78451.......... .......... .......... .......... ..........         >L78453.......... .......... .......... .......... ..........301                                                350 NC_001463 (gag)ATGTGTTGCC AAATGGGGAT GAGACCTGAG ACATTGCAAG ATGCAATGGC         >L78446.......... .......... .......... .......... ..........         >L78447.......... .......... .......... .......... ..........         >L78450.......... .......... .......... .......... ..........         >L78451.......... .......... .......... .......... ..........         >L78453.......... .......... .......... .......... ..........351                                                400 NC_001463 (gag)TACAGTAATC ATGAAAGATG GGTTACTGGA ACAAGAGGAA AAGAAGGAAG         >L78446.......... .......... .......... .......... ..........         >L78447.......... .......... .......... .......... ..........         >L78450.......... .......... .......... .......... ..........         >L78451.......... .......... .......... .......... ..........         >L78453.......... .......... .......... .......... ..........401                                                450 NC_001463 (gag)ACAAAAGAGA AAAGGAAGAG AGTGTCTTCC CAATAGTAGT GCAAGCAGCA         >L78446.......... .......... .......... .......... ..........         >L78447.......... .......... .......... .......... ..........         >L78450.......... .......... .......... .......... ..........         >L78451.......... .......... .......... .......... ..........         >L78453.......... .......... .......... .......... ..........451                                                500 NC_001463 (gag)GGAGGGAGAA GCTGGAAAGC AGTAGAITCT GTAATGTTCC AGCAACTGCA         >L78446.......... .......... .......... .......... ..........         >L78447.......... .......... .......... .......... ..........         >L78450.......... .......... .......... .......... ..........         >L78451.......... .......... .......... .......... ..........         >L78453.......... .......... .......... .......... ..........501                                                550 NC_001463 (gag)AACAGTAGCA ATGCAGCATG GCCTCGTGTC TGAGGACTTT GAAAGGCAGT         >L78446.......... ...CAGCATG GCCTCGTGTC CGAGGACTTT GAAAGGCAGT         >L78447.......... ...CAGCATG GAATAGTATC AGAAGAGTTT GAGAGGCAAC         >L78450.......... ...CAACATG GGATAGTATC AGAGGAATTT GAGAGACAAA         >L78451.......... ...CAGCATG GACTAGTATC AGAAGAATTT GAAAGGCAGC         >L78453.......... ...CAGCATG GACTTGTGTC CGAAGATTTT GAGAGGCAAT551                                                600 NC_001463 (gag)TGGCATATTA TGCTACTACC TGGACAAGTA AAGACATACT AGAAGTATTG         >L78446TGGCATATTA TGCTACTACC TGGACAAGTA AGGACATATT AGAAGTATTG         >L78447TGTCTTATTA TGCTACCACT TCGACAAGCA AGGATATCTT AGAGGTACTA         >L78450TGTCTTATTA TGCTACCACA TGGACAAGTA AGGATATTTT AGAAGTACTA         >L78451TAGCATACTA TGCCACAACG TGGACAAGCA AAGACATACT AGAGGTGTTA         >L78453TGGCATATTA TGCTACAACC TGGACTAGTG AAGATATATT AGAAGTATTG601                                                650 NC_001463 (gag)GCCATGATGC CTGGAAATAG AGCTCAAAAG GAGTTAATTC AAGGGAAATT         >L78446GCCATGATGC CAGGAAATAG AGCTCAAAAG GAGCTAATTC AA........         >L78447GCCATGATGC CTGGCAATAG AGCATTAAAA GAGCTAATAC AA........         >L78450GCAATCATGC CCGGGAACAG AGCATTAAAG GAGCTGATAC AA........         >L78451GCCATGATGC CAGGGAATAG AGCACAAAAA GAACTAATAC AA........         >L78453GCTATGATGC CTGGGAATAG AGCACAGAAA GAATTAATAC AA........651                                                700 NC_001463 (gag)AAATGAAGAA GCAGAAAGGT GGAGAAGGAA TAATCCACCA CCTCCAGCAG         >L78446.......... .......... .......... .......... ..........         >L78447.......... .......... .......... .......... ..........         >L78450.......... .......... .......... .......... ..........         >L78451.......... .......... .......... .......... ..........         >L78453.......... .......... .......... .......... ..........701                                                750 NC_001463 (gag)GAGGAGGATT AACAGTGGAT CAAATTATGG GGCTAGGACA AACAAATCAA         >L78446.......... .......... .......... .......... ..........         >L78447.......... .......... .......... .......... ..........         >L78450.......... .......... .......... .......... ..........         >L78451.......... .......... .......... .......... ..........         >L78453.......... .......... .......... .......... ..........751                                                800 NC_001463 (gag)GCAGCAGCAC AAGCTAACAT GGATCAGGCA AGGCAAATAT GCCTGCAATG         >L78446.......... .......... .......... .......... ..........         >L78447.......... .......... .......... .......... ..........         >L78450.......... .......... .......... .......... ..........         >L78451.......... .......... .......... .......... ..........         >L78453.......... .......... .......... .......... ..........801                                                850 NC_001463 (gag)GGTAATAAAT GCATTAAGAG CAGTAAGACA TATGGCGCAC AGGCCAGGGA         >L78446.......... .......... .......... .......... ..........         >L78447.......... .......... .......... .......... ..........         >L78450.......... .......... .......... .......... ..........         >L78451.......... .......... .......... .......... ..........         >L78453.......... .......... .......... .......... ..........851                                                900 NC_001463 (gag)ATCCAATGCT AGTAAAGCAA AAAACGAATG AGCCATATGA AGATTTTGCA         >L78446.......... .......... .......... .......... ..........         >L78447.......... .......... .......... .......... ..........         >L78450.......... .......... .......... .......... ..........         >L78451.......... .......... .......... .......... ..........         >L78453.......... .......... .......... .......... ..........901                                                950 NC_001463 (gag)CCAAGACTGC TAGAAGCAAT AGATOCAGAG CCAGTTACAC AGCCTATAAA         >L78446.......... .......... .......... .......... ..........         >L78447.......... .......... .......... .......... ..........         >L78450.......... .......... .......... .......... ..........         >L78451.......... .......... .......... .......... ..........         >L78453.......... .......... .......... .......... ..........951                                               1000 NC_001463 (gag)AGATTATCTA AAGCTAACAC TATCTTATAC AAATGCATCA GCAGATTGTC         >L78446.......... .......... .......... .......... ..........         >L78447.......... .......... .......... .......... ..........         >L78450.......... .......... .......... .......... ..........         >L78451.......... .......... .......... .......... ..........         >L78453.......... .......... .......... .......... ..........1001                                              1050 NC_001463 (gag)AGAAGCAAAT GGATAGAACA CTAGGACAAA GAGTACAACA AGCTAGTGTA         >L78446.......... .......... .......... .......... ..........         >L78447.......... .......... .......... .......... ..........         >L78450.......... .......... .......... .......... ..........         >L78451.......... .......... .......... .......... ..........         >L78453.......... .......... .......... .......... ..........1051                                              1100 NC_001463 (gag)GAAGAAAAAA TGCAAGCATG TAGAGATGTG GGATCAGAAG GGTTCAAAAT         >L78446.......... .......... .......... .......... ..........         >L78447.......... .......... .......... .......... ..........         >L78450.......... .......... .......... .......... ..........         >L78451.......... .......... .......... .......... ..........         >L78453.......... .......... .......... .......... ..........1101                                              1150 NC_001463 (gag)GCAATTGTTA GCACAAGCAT TAAGGCCAGG AAAAGGAAAA GGGAATGGAC         >L78446.......... .......... .......... .......... ..........         >L78447.......... .......... .......... .......... ..........         >L78450.......... .......... .......... .......... ..........         >L78451.......... .......... .......... .......... ..........         >L78453.......... .......... .......... .......... ..........1151                                              1200 NC_001463 (gag)AGCCACAAAG GTGTTACAAC TGTGGAAAAC CGGGACATCA AGCAAGGCAA         >L78446.......... .......... .......... .......... ..........         >L78447.......... .......... .......... .......... ..........         >L78450.......... .......... .......... .......... ..........         >L78451.......... .......... .......... .......... ..........         >L78453.......... .......... .......... .......... ..........1201                                              1250 NC_001463 (gag)TGTAGACAAG GAATCATATG TCACAACTGT GGAAAGAGAG GACATATGCA         >L78446.......... .......... .......... .......... ..........         >L78447.......... .......... .......... .......... ..........         >L78450.......... .......... .......... .......... ..........         >L78451.......... .......... .......... .......... ..........         >L78453.......... .......... .......... .......... ..........1251                                              1300 NC_001463 (gag)AAAAGAATGC AGAGGAAAGA GAGACATAAG GGGAAAACAG CAGGGAAACG         >L78446.......... .......... .......... .......... ..........         >L78447.......... .......... .......... .......... ..........         >L78450.......... .......... .......... .......... ..........         >L78451.......... .......... .......... .......... ..........         >L78453.......... .......... .......... .......... ..........1301                                           1347 NC_001463 (gag)GGAGGAGGGG GATACGTGTG GTGCCGTCCG CTCCTCCTAT GCAATAA         >L78446.......... .......... .......... .......... .......         >L78447.......... .......... .......... .......... .......         >L78450.......... .......... .......... .......... .......         >L78451.......... .......... .......... .......... .......         >L78453.......... .......... .......... .......... .......

TABLE 14 Tables for alignment of gag sequences NC_001463(gag720bp) vs.AF015181 Positives: 41.0% Identity: 41.0% NC_001463 (gag720bp) >AF015181NC_001463(gag720bp) 100 41 >AF015181 100 NC_001463(gag) vs. AF015181Positives: 40.6% Identity: 40.6% NC_001463 (gag) >AF015181NC_001463(gag) 100 41 >AF015181 100 NC_001463(gag 720bp) vs. AF402664˜8Positives: 91.1% Identity: 32.2% NC_001463(gag720bp) >AF402664 >AF402665 >AF402666 >AF402667 >AF402668NC_001463(gag720bp) 100 43 44 43 33 43 >AF402664 100 99 96 8098 >AF402665 100 97 80 99 >AF402666 100 81 96 >AF402667 100 80 >AF402668100 NC_001463(gag) vs. AF402664˜8 Positives: 49.1% Identity: 35.0%NC_001463 (gag) >AF402664 >AF402665 >AF402666 >AF402667 >AF402668NC_001463(gag) 100 45 46 44 36 45 >AF402664 100 99 98 89 99 >AF402665100 98 89 99 >AF402666 100 90 98 >AF402667 100 89 >AF402668 100NC_001463(gag720bp) vs. AJ305040˜2 Positives: 80.5% Identity: 38.1%NC_001463 (gag720bp) >AJ305040 >AJ305041 >AJ305042 NC_001463(gag720bp)100 39 42 39 >AJ305040 100 93 99 >AJ305041 100 93 >AJ305042 100NC_001463(gag) vs. AJ305040˜2 Positives: 44.3% Identity: 38.8% NC_001463(gag) >AJ305040 >AJ305041 >AJ305042 NC_001463(gag) 100 41 4140 >AJ305040 100 96 100 >AJ305041 100 96 >AJ305042 100NC_001463(gag720bp) vs. AY047362 Positives: 40.2% Identity: 40.2%NC_001463 (gag720bp) >AY047362 NC_001463(gag720bp) 100 40 >AY047362 100NC_001463(gag) vs. AY047362 Positives: 35.7% Identity: 35.7% NC_001463(gag) >AY047362 NC_001463(gag) 100 36 >AY047362 100 NC_001463(gag720bp)vs. AY081139 Positives: 40.0% Identity: 40.0% NC_001463(gag720bp) >AY081139 NC_001463(gag720bp) 100 40 >AY081139 100NC_001463(gag) vs. AY081139 Positives: 39.8% Identity: 39.8% NC_001463(gag) >AY081139 NC_001463(gag) 100 40 >AY081139 100 NC_001463(gag720bp)vs. AY101347˜8 Positives: 78.1% Identity: 35.0% NC_001463(gag720bp) >AY101347 >AY101348 NC_001463(gag720bp) 100 40 36 >AY101347100 91 >AY101348 100 NC_001463(gag) vs. AY101347˜8 Positives: 43.9%Identity: 37.9% NC_001463 (gag) >AY101347 >AY101348 NC_001463(gag) 10041 40 >AY101347 100 95 >AY101348 100 NC_001463(gag720bp) vs. L78446, 7,50, 51, 53 Positives: 17.6% Identity: 11.9% NC_001463(gag720bp) >L78446 >L78447 >L78450 >L78451 >L78453 NC_001463(gag720bp)100 17 14 14 15 15 >L78446 100 96 96 97 97 >L78447 100 98 97 96 >L78450100 96 96 >L78451 100 97 >L78453 100 NC_001463(gag) vs. L78446, 47, 50,51, 53 Positives: 9.4% Identity: 6.4% NC_001463(gag) >L78446 >L78447 >L78450 >L78451 >L78453 NC_001463(gag) 100 9 7 7 88 >L78446 100 98 98 98 98 >L78447 100 99 98 98 >L78450 100 98 98 >L78451100 98 >L78453 100

TABLE 15 NC_001463(full genome) vs.AF322109(full genome) Positives:68.2% Identity: 68.2% NC_001463 AF322109 NC_001463 100  68 AF322109 100NC_001463(gag) vs.AF322109(gag) Positives: 73.1% Identity: 73.1%NC_001463 AF322109 (gag) (gag) NC_001463(gag) 100  73 AF322109(gag) 100NC_001463(5′LTR region) vs.AF322109(5′LTR region) Positives: 59.8%Identity: 59.8% NC_001463 AF322109 (5′) (5′) NC_001463(5′) 100  60AF322109(5′) 100 NC_001463(pol) vs.AF322109(pol) Positives: 74.9%Identity: 74.9% NC_001463 AF322109 (pol) (pol) NC_001463(pol) 100  75AF322109(pol) 100 NC_001463(rev) vs.AF322109(rev) Positives: 48.3%Identity: 48.3% NC_001463 AF322109 (rev) (rev) NC_001463(rev) 100  48AF322109(rev) 100 NC_001463(vif) vs.AF322109(vif) Positives: 66.0%Identity: 66.0% NC_001463 AF322109 (vif) (vif) NC_001463(vif) 100  66AF322109(vif) 100

1. A recombinant caprine arthritis encephalitis virus (CAEV)-basedtransfer vector, comprising: (a) A (CAEV) packaging sequence consistingessentially of (i) 5′ untranslated region consisting of nucleotides 1 to511 of the CAEV genome and (ii) nucleotides about 1 to X of the CAEVgag-encoding sequence linked to the 3′ end of the 5′ untranslatedregion, wherein X is less than about 613; and (b) cis-acting elementsoperably linked to the CAEV packaging sequence. 2-5. (canceled)
 6. Thetransfer vector of claim 1, wherein X is about
 327. 7. The transfervector of claim 1, wherein the start codon of the gag-encoding sequenceis mutated to prevent translation of gag protein.
 8. The transfer vectorof claim 7, wherein said start codon is mutated to TAG.
 9. The transfervector of claim 7, wherein the ATG codon of the gag-encoding sequencelocated 21 base pairs downstream of the start codon ATG is mutated toprevent translation of gag protein.
 10. (cancelled)
 11. The transfervector of claim 1, wherein the cis-acting elements comprise one or moresequences selected from the group consisting of RRE (rev-responsiveelement) region, CAEV 3′ LTR of which U3 region is deleted, and aheterologous promoter.
 12. (cancelled)
 13. (cancelled)
 14. The transfervector of claim 11, wherein the heterologous promoter is the humancytomegalovirus major immediate early promoter (HCMV MIEP).
 15. Thetransfer vector of claim 1, wherein said vector has the structure ofpCAH/SINd1 shown in FIG. 3C.
 16. The transfer vector of claim 1, whichfurther comprises a transcription cassette comprising a heterologousgene operably linked to a heterologous promoter.
 17. (cancelled)
 18. Arecombinant CAEV-based vector system comprising the transfer vector ofclaim 1, and a packaging vector system, wherein said packaging vectorsystem comprises: a first polynucleotide comprising a CAEVgag-pol-encoding sequence and an RRE, and a second polynucleotidecomprising a viral envelope-encoding sequence.
 19. The vector system ofclaim 18, wherein said transfer vector further comprises a transcriptioncassette comprising a heterologous gene operably linked to aheterologous promoter.
 20. (cancelled)
 21. The vector system of claim18, wherein said viral envelope-encoding sequence is a non-CAEVenvelope-encoding sequence.
 22. The vector system of claim 21, whereinsaid non-CAEV envelope-encoding sequence is VSV-G (vesicular somatitisvirus G) glycoprotein-encoding sequence or GaLV (gibbon ape leukemiavirus) envelope protein-encoding sequence.
 23. (cancelled) 24.(cancelled)
 25. The vector system of claim 18, wherein said vectorsystem further comprises a third polynucleotide sequence comprising arev-encoding sequence.
 26. The vector system of claim 18, wherein saidvector system further comprises a fourth polynucleotide sequencecomprising a vif-encoding sequence. 27.-32. (canceled)
 33. The vectorsystem of claim 18, wherein said packaging vector system is devoid of acompetent CAEV packaging sequence.
 34. (cancelled)
 35. The vector systemof claim 18, which comprises a first vector comprising said firstpolynucleotide and a second vector comprising said secondpolynucleotide. 36.-51. (canceled)
 52. A method for delivering apolypeptide into a mammalian cell comprising contacting said mammaliancell with replication-defective vector particles prepared bytransfecting a cell with the recombinant CAEV-based vector system ofclaim
 19. 53.-56. (canceled)
 57. A method for delivering a polypeptideinto a vertebrate comprising administering to the vertebratereplication-defective vector particles prepared by transfecting a cellwith the recombinant CAEV-based vector system of claim
 19. 58.-72.(canceled)
 73. The transfer vector of claim 15, wherein said vector isat least 70% identical to SEQ ID NO:
 68. 74.-78. (canceled)